WO2023138661A1 - Nkg2a antibody and application thereof - Google Patents

Nkg2a antibody and application thereof Download PDF

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Publication number
WO2023138661A1
WO2023138661A1 PCT/CN2023/073176 CN2023073176W WO2023138661A1 WO 2023138661 A1 WO2023138661 A1 WO 2023138661A1 CN 2023073176 W CN2023073176 W CN 2023073176W WO 2023138661 A1 WO2023138661 A1 WO 2023138661A1
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antibody
seq
cells
nkg2a
amino acid
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French (fr)
Chinese (zh)
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WO2023138661A9 (en
WO2023138661A8 (en
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周亮
王鹏
李宗海
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恺兴生命科技(上海)有限公司
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Publication of WO2023138661A9 publication Critical patent/WO2023138661A9/en
Publication of WO2023138661A8 publication Critical patent/WO2023138661A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of tumor immunotherapy or diagnosis, more specifically, to an antibody recognizing NKG2A and its application.
  • NK cells Natural killer cells
  • NK cells are a type of lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization, and are one of the important components of the body's immune cells.
  • receptors on the surface of NK cells which are divided into two types: activating and inhibitory, and NKG2A is one of the inhibitory receptors.
  • NKG2A protein also called C-type lectin, is expressed not only in NK cells, but also in NKT cells and CD8+ ⁇ T cells. NKG2A can form dimers with CD94 on the cell surface (Jiacheng Bi and Zhigang Tian. NK Cell Dysfunction and Checkpoint Immunotherapy. Front. Immunol, 2019.).
  • the non-classical MHC class I molecule HLA-E is the main ligand of NKG2A-CD94. Under normal circumstances, the expression level of HLA-E is very low, but on the surface of most tumor cells, the expression level of HLA-E increases.
  • NKG2A and HLA-E inhibit the activation of NK cells, making tumor cells avoid being killed by NK cells (Linda Borst, et al.
  • NKG2A antibodies have great potential, their development is extremely challenging.
  • the NKG2 receptor family has a variety of receptors, including NKG2A, NKG2C, NKG2D, and NKG2E, among others. Some of these receptors have an inhibitory effect on immune cells, and some have an activating effect.
  • the amino acid sequences of these receptors have high homology, among which the homology of human NKG2A and NKG2C is 90%, and that of NKG2E is 71%.
  • NKG2C and NKG2E are very similar to NKG2A in the extracellular sequence, they are completely opposite in function and require high antibody specificity.
  • NKG2A target monoclonal antibody drugs have been reported, mainly including Innate/AstraZeneca’s Monalizumab (US20170298131A1) and Huaiyue Biotech’s Mpb416 (CN111153995A).
  • Preliminary clinical results show that the combination of NKG2A antibodies has a certain anti-tumor effect, but there are also problems of poor single-drug effect and certain side effects.
  • the purpose of the present invention is to provide a fully human antibody recognizing NKG2A and its application.
  • the present invention provides a fully human antibody that recognizes NKG2A, wherein the antibody comprises a light chain variable region, and the light chain variable region comprises LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYX 1 X 2 T (SEQ ID NO:129), wherein X 1 is I or V, X2 is R or S.
  • the antibody comprises a light chain variable region, and the light chain variable region comprises LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYX 1 X 2 T (SEQ ID NO:129), wherein X 1 is I or V, X2 is R or S.
  • the antibody comprises a light chain variable region comprising LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYIRT (SEQ ID NO:6).
  • the antibody comprises a light chain variable region comprising LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYVST (SEQ ID NO:10).
  • the present invention also provides a fully human antibody that recognizes NKG2A, characterized in that the antibody includes a heavy chain variable region, and the heavy chain variable region is selected from:
  • HCDR1 comprising X 1 X 2 X 3 X 4 S (SEQ ID NO: 131), wherein X 1 is S, R or N, X 2 is Y, F or V, X 3 is A, Y or H, X 4 is M or V; and/or HCDR2 shown in AIX 1 X 2 X 3 X 4 GSTYYADSVKG (SEQ ID NO: 132), wherein X 1 is S, T Or N, X2 is G or A, X3 is S, W, G or P, X4 is G or V; and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  • the antibody comprises a heavy chain variable region comprising HCDR1 shown in SYAIS (SEQ ID NO:1); and/or HCDR2 shown in GIIPIFGTANYAQKFQG (SEQ ID NO:2); and/or HCDR3 shown in GFDGMDY (SEQ ID NO:3).
  • the antibody comprises a heavy chain variable region comprising HCDR1 shown in SYAIS (SEQ ID NO:1); and/or HCDR2 shown in GIIPIFGTAHYAQKFQG (SEQ ID NO:11); and/or HCDR3 shown in GFDGMDY (SEQ ID NO:3).
  • the antibody comprises a heavy chain variable region comprising HCDR1 shown in SYAMS (SEQ ID NO:7); and/or HCDR2 shown in AISGSGGSTYYADSVKG (SEQ ID NO:8); and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  • the antibody comprises a heavy chain variable region comprising HCDR1 shown in RFYMS (SEQ ID NO: 12); and/or HCDR2 shown in AITGWGGSTYYADSVKG (SEQ ID NO: 13); and/or HCDR3 shown in GYDGFDY (SEQ ID NO: 9).
  • the antibody comprises a heavy chain variable region comprising HCDR1 shown in RVHMS (SEQ ID NO: 14); and/or HCDR2 shown in AISAGGGSTYYADSVKG (SEQ ID NO: 15); and/or HCDR3 represented by GYDGFDY (SEQ ID NO:9).
  • the antibody comprises a heavy chain variable region comprising HCDR1 shown in NFHVS (SEQ ID NO: 16); and/or HCDR2 shown in AINGPVGSTYYADSVKG (SEQ ID NO: 17); and/or HCDR3 shown in GYDGFDY (SEQ ID NO: 9).
  • the antibody is selected from any of the following:
  • an antibody comprising a heavy chain variable region, the heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising HCDR2 shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and/or comprising HCDR3 shown in any of SEQ ID NO: 3 or 9;
  • an antibody comprising a light chain variable region comprising LCDR1 shown in SEQ ID NO:4, and/or comprising LCDR2 shown in SEQ ID NO:5, and/or comprising LCDR3 shown in any of SEQ ID NO:6 or 10;
  • an antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
  • the antibody is selected from any of the following:
  • an antibody comprising HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:10; or
  • the antibody is selected from any of the following:
  • an antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or a variant of the above sequence;
  • an antibody comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence;
  • An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody.
  • the antibody is selected from any of the following:
  • an antibody the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:20;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24;
  • the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24.
  • the antibody described in the first aspect is a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or IgG4 antibody.
  • the antibody of the first aspect does not significantly bind NKG2C, NKG2E or a combination thereof.
  • the antibody of the first aspect binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94 or a combination thereof.
  • the antibody of the first aspect binds to cells expressing NKG2A/CD94, but does not significantly bind to cells expressing NKG2C/CD94, NKG2E/CD94 or a combination thereof.
  • the antibody described in the first aspect is more effective in reducing CD94/NKG2A-mediated cytotoxicity of CD94/NKG2A-expressing cytotoxic lymphocytes.
  • the CD94/NKG2A-expressing cytotoxic lymphocytes are NK cells, NKT cells, ⁇ / ⁇ T cells or ⁇ / ⁇ T cells.
  • said CD94/NKG2A expressing cytotoxic lymphocytes are NK cells.
  • the present invention provides an immunoconjugate, which includes the anti- body, and functional molecules linked to it.
  • the present invention provides a chimeric receptor, the ectodomain of the chimeric receptor comprises the antibody described in the first aspect, and the chimeric receptor includes: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC) or a combination thereof.
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • said chimeric receptor is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the CAR comprises sequentially connected: the antibody described in the first aspect, a transmembrane region and an intracellular signal region.
  • the intracellular signal region is selected from: the intracellular signal region sequence of CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, CD40 or a combination thereof; and/or the transmembrane region comprises the transmembrane region of CD8 or CD28.
  • the CAR includes: the antibody of the first aspect, the transmembrane region of CD8/CD28, and CD3 ⁇ ; or the antibody of the first aspect, the transmembrane region of CD8/CD28, the intracellular signal region of CD137, and CD3 ⁇ ; or the antibody of the first aspect, the transmembrane region of CD8/CD28, the intracellular signal region of CD28, and CD3 ⁇ ; or the antibody of the first aspect, the transmembrane region of CD8/CD28, and the intracellular signal region of CD28 , CD137 and CD3 ⁇ .
  • amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 115 or 116.
  • the present invention provides nucleic acid encoding the antibody of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect.
  • the present invention provides an expression vector comprising the nucleic acid described in the fourth aspect.
  • the present invention provides a virus comprising the expression vector of the fifth aspect or the nucleic acid of the fourth aspect.
  • the nucleic acids, expression vectors and viruses involved in the fourth, fifth and sixth aspects are all biological materials of the present invention.
  • the biological material of the present invention is any one of the following:
  • the present invention provides a host cell expressing the chimeric receptor of the third aspect.
  • the host cell binds to cells expressing NKG2A/CD94 and does not significantly bind NKG2C/CD94, NKG2E/CD94, or a combination thereof.
  • the host cell can resist NK cell attack or kill NK cell.
  • the host cell also expresses a chimeric receptor that recognizes tumor antigens and/or pathogen antigens.
  • said host cell is used in combination with a second host cell targeting a tumor and/or a pathogen.
  • the host cell and/or the second host cell does not express B2M, TCR/B2M, TCR/B2M/CIITA, TCR/B2M/NKG2A, and/or TCR/B2M/CIIA/NKG2A.
  • the host cell and/or the second host cell is a T cell, a natural killer cell, a cell Toxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, stem cell-derived immune effector cells, or combinations thereof.
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells.
  • said T cells are autologous/allogeneic T cells.
  • said T cells are primary T cells.
  • the T cells are derived from human T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), ⁇ T cells or combinations thereof.
  • the present invention provides a drug combination, the antibody described in the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, and the host cell described in the seventh aspect are administered in combination with agents that enhance their functions, preferably, in combination with chemotherapy drugs; and/or in combination with agents that improve one or more side effects associated therewith; and/or in combination with host cells expressing chimeric antigen receptors targeting NKG2A.
  • the present invention provides a method for preparing the antibody of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect, the method comprising culturing the host cell of the seventh aspect under conditions suitable for expressing the antibody, immunoconjugate, and chimeric receptor, and isolating the antibody, immunoconjugate, composition, and/or chimeric receptor expressed by the host cell.
  • the present invention provides a pharmaceutical composition, which includes: the antibody described in the first aspect or the nucleic acid encoding the antibody; or the immunoconjugate described in the second aspect or the nucleic acid encoding the conjugate; or the chimeric receptor described in the third aspect or the nucleic acid encoding the chimeric receptor; or the host cell described in the seventh aspect; and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a kit comprising:
  • Figure 1 shows a schematic diagram of constructing vectors expressing NKG2A/CD94 and NKG2C/CD94 heterodimers respectively using the eukaryotic expression plasmid V152S;
  • Figure 2 shows the binding of ELISA detection antibodies A1, A2 (Fab form) to NKG2A/CD94, NKG2C/CD94;
  • Figure 3 shows the EC50 of ELISA detection antibody A1, A2 (IgG4 form) binding to NKG2A/CD94;
  • Fig. 4 shows the affinity of Biacore detection antibody A1, A2 (IgG4 form);
  • Figure 5 shows the vector map of the target gene CD94-Flag, NKG2A-CD94-Flag, NKG2C-CD94-Flag and NKG2E-CD94-Flag with Flag;
  • FIG. 6 shows the binding of FACs detection antibodies A1, A2 (IgG4 form) to CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94, CHOK1-NKG2E-CD94 and CHOK1-CD94 cells;
  • Figure 7 shows the amino acid sequence comparison of the heavy chain variable regions of antibodies A1, A2, A3, A4, A5, and A6;
  • FIG. 8 shows the binding of FACs detection antibodies A1, A2, A3, A4, A5, A6 (Fab form) to CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94 and CHOK1-NKG2E-CD94 cells;
  • Figure 9 shows the EC50 of ELISA detection antibodies A1, A2, A3, A4, A5, A6 (Fab format) combined with NKG2A/CD94 heterodimer;
  • Figure 10 shows the EC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (Fab form) combined with CHO-K1 cells overexpressing NKG2A/CD94 heterodimer;
  • Figure 11 shows the EC50 of ELISA detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) combined with NKG2A/CD94 heterodimer;
  • Figure 12 shows the EC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) and CHOK1 cells overexpressing NKG2A/CD94;
  • FIG 13 shows the affinity of Biacore detection antibodies A3, A4, A5, A6 (IgG4 form);
  • Figure 14 shows the vector maps of pET22b-HLA-E and pET22b- ⁇ 2m
  • Figure 15 shows the EC50 of HLA-E tetramer binding to NK cells expressing NKG2A/CD94;
  • Figure 16 shows the IC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) blocking the combination of NKG2A and its ligand HLA-E;
  • Figure 17 shows the expression levels of HLA-E on tumor cells K562-HLA-E, K562 and FaDu cells;
  • Figure 18 shows the expression of CD107a detected by FACs after the co-incubation of NK cells with K562, K562-HLA-E, FaDu cells and the addition of antibodies A1, A2, A3, A4, A5, A6 (IgG4 form);
  • Figure 19 shows the effects of FACs detection of anti-NKG2A antibodies on the killing effect of NK cells on K562, K562-HLA-E, and FaDu cells;
  • Figure 20 shows the changes in the proportion of NK cells after co-incubation of A4-BBZ CAR T cells, A5-BBZ CAR T cells and NK cells.
  • Figure 21 shows the changes in the ratio of UCAR-T cells after co-incubation of A4 and A5 UCAR-T cells with NK cells.
  • Figure 22 shows the joint action of A4, A5-UCAR-T and BCMA UCAR-T in the presence of NK cells for subcutaneous xenografts of RPMI-8226 cells.
  • the inventors After in-depth research and screening, the inventors obtained a fully human antibody specifically recognizing NKG2A, including the Fab form Antibodies in IgG4 form.
  • the antibody of the present invention can be applied to the preparation of targeted anti-tumor drugs and drugs for diagnosing tumors.
  • range format various aspects of claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual values within that range. For example, where a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range, as well as any other stated or intervening value within the range, is encompassed within the claimed subject matter, and that the upper and lower limits of the range are also within the scope of the claimed subject matter.
  • any concentration range, percentage range, ratio range or integer range recited herein is to be understood as including any integer and, where appropriate, fractional values (eg, tenths and hundredths of an integer) thereof within the stated range.
  • NKG2A Natural killer group 2A
  • KLRC1 natural killer cell lectin like receptor C1
  • CD159a CD159a
  • NK cell receptor A is one of the transmembrane proteins preferentially expressed on the surface of NK cells.
  • NKG2A is mainly expressed on the surface of NK cells and some T cells (CD8+T cells, Th2 cells, ⁇ T cells and NKT cells).
  • the heterodimer NKG2A-CD94 formed by NKG2A and CD94 molecules (NK cell surface membrane protein) in the form of disulfide bonds is recognized by the non-classical histocompatibility complex I (major histocompatibility complex class I, MHC I) molecule HLA-E on the target cells.
  • the expression of this molecule is low under normal conditions, but on the surface of most tumor cells, the expression of HLA-E increases , thereby inducing a cascade of inhibitory signals, inhibiting the cytotoxic activity of NK and the secretion of cytokines. Certain viral infections, tumors and immune diseases escape immune inspection through this pathway.
  • NKG2A is highly expressed in NK cells activated by IL15.
  • the NKG2 family also includes NKG2C, NKG2D and NKG2E.
  • NKG2A includes any native NKG2A from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats).
  • the term includes "full length” unprocessed NKG2A as well as any form of NKG2A derived from processing in the cell.
  • the term also includes naturally occurring variants of NKG2A, such as splice variants or allelic variants.
  • the anti-NKG2A antibodies described herein inhibit the binding of NKG2A protein to HLA-E, thereby functioning as checkpoint inhibitors.
  • the amino acid sequence of the full-length human NKG2A is shown in SEQ ID NO:68
  • the amino acid sequence of the extracellular segment of NKG2A is shown in SEQ ID NO:70
  • the amino acid sequence of the full-length human NKG2C is shown in SEQ ID NO:123
  • the amino acid sequence of the extracellular segment of NKG2C is shown in SEQ ID NO:72
  • the amino acid sequence of the full-length human NKG2E is shown in SEQ ID NO:125.
  • the amino acid sequence of the extracellular segment of KG2E is shown in SEQ ID NO:74.
  • Human leukocyte antigen (Human leukocyte antigen, HLA) is the coding gene of the human major histocompatibility complex MHC, which is closely related to the function of the human immune system.
  • HLA includes class I, class II and class III gene portions.
  • the antigens expressed by HLA class I and class II genes are located on the cell membrane and are MHC-I (encoded by HLA-A, HLA-B, HLA-C sites) and MHC-II (encoded by HLA-D region).
  • HLA class I is distributed on the surface of almost all cells in the body. It is a heterodimer composed of heavy chain ( ⁇ chain) and ⁇ 2 microglobulin (B2M).
  • Class II is mainly located in macrophages Glycoproteins on the surface of cells and B lymphocytes.
  • HLA-E (OMIM 143010, gene number NM_005516.6) is a non-classical MHC molecule expressed on the cell surface and regulated by the binding of peptides such as fragments of signal sequences derived from other MHC class I molecules. HLA-E through specific binding CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C (see, for example, Braud and others, (1998) Nature 391: 795-799, all of which are combined with increasing this article by citing this article), and natural killing (NK) cells, natural killing killings) T cells (NKT) and sub -groups of T cells ( ⁇ / ⁇ and ⁇ / ⁇ ).
  • HLA-E refers to any variant, derivative or isoform of the HLA-E gene or encoded protein.
  • SEQ ID NO:78 The amino acid sequence of human HLA-E extracellular region is shown in SEQ ID NO:78. HLA-E is widely distributed in low-level cells throughout the body.
  • High levels of HLA-E are found in several tumors, including gynecologic tumors (up to 90% of tumor samples), and up to 50% of breast, non-small cell lung (NSCLC), liver, pancreas, kidney, melanoma, prostate, head and neck, stomach, rectal, and colorectal cancers.
  • NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment Clin Cancer Res.2020 Nov 1; 26(21):5549-5556.
  • Tumors with high HLA-E expression escape immune cell killing by combining with NKG2A of immune cells (such as NK cells and T cells).
  • the NKG2A antibody disclosed in the present invention can inhibit the immune escape of tumors with high HLA-E expression to kill the tumor cells.
  • lysosome-associated membrane protein 1 (CD107a) is the main component of vesicle membrane protein, and mainly constitutes cytotoxic granules in the form of vesicles in the cytoplasm of cells.
  • NK cells kill target cells, the released cytotoxic granules will reach the target cell membrane and fuse with the target cell membrane, causing the release of the granule contents, eventually leading to the death of the target cell.
  • the spontaneous expression rate of CD107a on the surface of NK cell membrane is very low, and the increase of CD107a expression can be detected on the surface after stimulation of target cells. Therefore, the increase of CD107a molecule after NK cell stimulation can reflect the level of cytotoxic cell killing activity of NK cells.
  • polypeptide peptide
  • protein protein
  • protein protein
  • polymer may be linear, cyclic or branched, it may comprise modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • amino acid polymers such as amino acid polymers that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenonylation, transfer-RNA-mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with a labeling component, etc.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes polypeptides expressed from a specified nucleic acid sequence.
  • antibody is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
  • Antibody fragment refers to a molecule other than an intact antibody that contains a fragment of the intact antibody in combination with the antigen to which the intact antibody binds. part.
  • antibody fragments include, but are not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F( ab') 2 fragments, bivalent fragments comprising 2 linked Fab fragments; (vi) antigen binding sites of single chain Fv molecules; (vii) bispecific single chain Fv dimers (PCT/US92/09965); (viii) "dimers” or “trimers", multivalent or multispecific fragments constructed by genetic
  • the "class" of an antibody refers to the type of constant domain or region that its heavy chain possesses.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • variable region or variable domain refers to the domains of an antibody heavy or light chain that participate in antibody antigen binding.
  • the heavy and light chain variable domains (VH and VL, respectively) of native antibodies typically have similar structures, with each domain comprising four conserved FRs and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., p. 91 (2007)).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind a particular antigen can be isolated using the VH or VL domains from antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • hypervariable region or “complementarity determining region” or “CDR” refers to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops") and/or contain residues that make contact with an antigen ("antigen contacts").
  • antibodies typically contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).
  • Fc region or “Fc” is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • FR Framework (FR) refers to variable domain residues that are distinct from hypervariable region (CDR) residues.
  • the FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3 and FR4.
  • CDR residues and other residues in the variable domain are numbered according to Kabat et al. above.
  • natural antibody refers to naturally occurring immunoglobulin molecules of various structures.
  • native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3).
  • VH variable region
  • CH1 variable heavy domain
  • CH3 constant domains
  • each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a light chain constant (CL) domain.
  • VH variable heavy domain
  • VL variable region
  • CL light chain constant domain
  • the light chains of antibodies can be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of their constant domains.
  • full antibody full-length antibody
  • intact antibody refers to a protein having a structure substantially similar to a natural antibody.
  • the structure either has a heavy chain comprising an Fc region as defined herein or comprises a complete full length antibody with an antigen binding region.
  • single domain antibody refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called a nanobody (Nanobody).
  • single domain antibody refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain.
  • the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Patent No. 6248516).
  • mAb refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during the preparation of monoclonal antibody preparations, such variants usually being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the designation "monoclonal” indicates that the nature of the antibody is that it was obtained from a substantially homogeneous population of antibodies, and is not considered to require that the antibody be prepared by any particular method.
  • they can be produced by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain of an antibody is derived from a particular source or species, and the remaining portion of the heavy and/or light chain is derived from a different source or species.
  • chimeric antibodies comprise non-human variable regions (eg, variable regions derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and human constant regions.
  • chimeric antibodies are "class-switched" antibodies, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • chimeric antibodies are "humanized antibodies.”
  • humanized is used for non-human antibodies, such as rodents or primates, etc., that are hybrid immunoglobulins, immunoglobulin chains or fragments thereof that contain minimal sequence derived from non-human immunoglobulins.
  • a “humanized antibody” refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one (typically two) variable domains in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized antibody” may include mutations, for example, introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
  • fully human antibody is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using human antibody repertoires or other human antibody coding sequences.
  • the definition of fully human specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • the antibodies provided herein are "fully human antibodies” generated by phage display technology.
  • Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies possessing the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with desired binding properties. library. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J .Mol.Biol.222:581-597 (1992); Marks, Meth.Mol.Biol., 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J.Mol.Biol.338(2):299-310 (2004); Lee
  • VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994).
  • Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas.
  • natural Libraries eg, from humans
  • the natural Libraries thus provide a single source of antibodies to multiple non-self as well as self antigens without requiring any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993).
  • natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and effecting the rearrangement in vitro as described by Hoogenboom, J. Mol. Biol. 227:381-388 (1992).
  • antibodies or antibody fragments isolated from fully human antibody libraries are considered fully human antibodies or fully human antibody fragments.
  • parent antibody refers to the antibody provided in this application or the antibody obtained after undergoing mutation or affinity maturation according to the antibody provided in this application.
  • the parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody.
  • a parent antibody may refer to the antibody itself, a composition comprising said parent antibody, or an amino acid sequence encoding it.
  • affinity matured antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to the parent antibody, such alterations resulting in an increase in the affinity of the antibody for the antigen.
  • HVRs hypervariable regions
  • variant refers to a polypeptide having substantially the same amino acid sequence or one or more activities encoded by the substantially same nucleotide sequence as the sequence of the antibody provided herein.
  • the variant has the same or similar activity as the antibody provided in the examples of the present application.
  • variant antibody or “antibody variant” includes antibody sequences that differ from a parent antibody sequence by at least one amino acid modification compared to the parent.
  • the variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity to the parent antibody sequence.
  • Antibody variants may refer to the antibody itself or to a composition comprising the antibody variant.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis.
  • amino acid modification includes amino acid substitution, addition and/or deletion
  • amino acid substitution and “amino acid replacement” mean replacing an amino acid at a specific position in a parental polypeptide sequence with another amino acid
  • amino acid insertion means adding an amino acid at a specific position in a parental polypeptide sequence
  • amino acid deletion means removing a specific position in a parental polypeptide sequence amino acids on. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct provided that the final construct possesses the desired characteristics, eg binding of antigen.
  • Modification refers to a change in the state or structure of the protein or polypeptide of the present invention. Modifications can be chemical, structural and functional.
  • conservative modification or “conservative sequence modification” means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis. Families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.
  • amino acid residues in the CDR regions or in the framework regions of an antibody of the invention can be replaced with other amino acid residues of the same side chain family, and the altered antibody (variant antibody) can be tested for retained function.
  • Non-conservative substitutions entail exchanging a member of one of these groups for a member of another group.
  • a substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody).
  • a parent antibody eg., a humanized or human antibody
  • the resulting variant selected for further study will have an altered (e.g., improved) certain biological property (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody.
  • exemplary substitutional variants are affinity matured antibodies, which can be routinely prepared, eg, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for specific biological activity (eg, binding affinity).
  • Alterations can be made in the CDR regions, eg, to increase antibody affinity.
  • Such alterations can be made in HVR "hotspots", i.e., codon-encoded residues that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008), and/or residues that relieve antigens, and the resulting variant VH or VL tested for binding affinity.
  • Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in HVR.
  • affinity maturation diversity is introduced into the variable genetic species selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Secondary levels are then generated library. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized.
  • substitutions, insertions, or deletions may occur within one or more CDRs, so long as such alterations do not significantly reduce the ability of the antibody to bind antigen.
  • conservative changes that do not significantly reduce binding affinity eg, the conservative modifications described herein
  • Such alterations may be, for example, outside of the residues in the CDRs that contact the antigen.
  • each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.
  • anti-NKG2A antibody refers to an antibody capable of binding NKG2A with sufficient affinity, which is useful as a diagnostic and/or therapeutic agent for targeting NKG2A.
  • the anti-NKG2A antibody binds to an irrelevant, non-NKG2A protein to an extent that is less than about 10% of the antibody's binding to NKG2A, as determined by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • an anti-NKG2A antibody binds an epitope of NKG2A that is conserved among NKG2A derived from different species.
  • antigen-binding proteins having a Fab (fragment of antigen-binding)-based antigen-binding region are described, including antibody Fab and antibody IgG4.
  • the NKG2A/CD94 heterodimer was used to select Fab from a fully human native Fab phage library. These molecules exhibit fine specificity.
  • the antibody only recognizes NKG2A, and does not recognize NKG2C and NKG2E.
  • NKG2A refers to human NKG2A.
  • the invention includes antibodies having a Fab, IgG4 sequence fused to one or more heavy chain constant regions to form an antibody with a human immunoglobulin Fc region to generate a bivalent protein, thereby increasing the overall affinity and stability of the antibody.
  • the Fc moiety allows direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to antibodies for example in antigen quantification studies, for immobilization of antibodies for affinity measurements, for targeted delivery of therapeutics, testing of Fc-mediated cytotoxicity using immune effector cells, and many other applications.
  • Antibodies or antibody fragments of the invention are based on the use of phage display to identify and select antigen-binding fragments (Fabs) whose amino acid sequence confers specificity to the antibody or antibody fragment against NKG2A and forms the basis of all antigen-binding proteins of the disclosure.
  • the Fab can be used to design a range of different "antibodies or antibody fragments" including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, IgG4, multivalent antibodies, i.e., antibodies with more than one specificity for the same antigen or different antigens, e.g., bispecific T cell-binding antibodies (BiTEs), tribodies, etc. (see Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotech Nology 28:355-362, 2010).
  • the invention provides full-length antibodies, the heavy and light chains of which can be full-length (e.g., an antibody can include at least one, preferably two intact heavy chains, and at least one, preferably two intact light chains) or can include an antigen-binding portion (Fab, F(ab')2, Fv, or scFv).
  • the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE. The choice of antibody type will depend on the immune effector function the antibody is designed to elicit.
  • the appropriate amino acid sequences for the constant regions of the various immunoglobulin isotypes and methods for producing the broad class of antibodies are known to those skilled in the art.
  • the present invention provides a fully human antibody that recognizes NKG2A, said antibody comprising a light chain variable region comprising LCDR1 represented by RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 represented by DASSLES (SEQ ID NO:5); and/or LCDR3 represented by QQYDSYX 1 X 2 T, wherein X 1 is selected from I, V, G, A, L, F or P, preferably, X 1 is selected from I or V X2 is selected from R, S, K, H, W, Y, C, M, N, Q or T, preferably, X2 is selected from R or S.
  • RASQSISSWLA SEQ ID NO:4
  • LCDR2 represented by DASSLES (SEQ ID NO:5)
  • LCDR3 represented by QQYDSYX 1 X 2 T
  • the present invention provides a fully human antibody that recognizes NKG2A, the antibody includes a heavy chain variable region, and the heavy chain variable region is selected from:
  • HCDR1 shown in SYAIS SEQ ID NO: 1
  • HCDR2 shown in GIIPIFGTAX 1 YAQKFQG (SEQ ID NO: 130)
  • X 1 is selected from N, H, K, R, W, Y, S, C, M, Q or T, preferably, X 1 is selected from N or H
  • HCDR3 shown in GFDGMDY (SEQ ID NO: 3);
  • HCDR1 represented by X 1 X 2 X 3 X 4 S (SEQ ID NO: 131); and/or HCDR2 represented by AIX 1 X 2 X 3 X 4 GSTYYADSVKG (SEQ ID NO: 132); and/or HCDR3 represented by GYDGFDY (SEQ ID NO: 9).
  • amino acids at the X 1 X 2 X 3 X 4 position in HCDR1 and HCDR2 are selected from Table 2 below.
  • the antibody further includes a framework region, wherein the 30th amino acid of the first framework region is selected from S, R, N, W, Y, C, M, Q, T, H, K, G, A, V, L, I, P, or F, preferably, selected from S, R, N or G.
  • the present invention provides an antibody that recognizes NKG2A, said antibody comprising a heavy chain variable region, said heavy chain variable region comprising heavy chain CDR1 of any amino acid sequence shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising heavy chain CDR2 of any amino acid sequence shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and/or comprising SEQ ID NO: 3 or 9 Heavy chain CDR3 of any amino acid sequence shown.
  • the present invention provides an antibody recognizing NKG2A comprising: light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, and/or light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and/or light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10.
  • the present invention provides an antibody recognizing NKG2A comprising: heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO:1, 7, 12, 14 or 16, and/or heavy chain CDR2 comprising any amino acid sequence shown in SEQ ID NO:2, 8, 11, 13, 15 or 17, and/or comprising heavy chain CDR3 of any amino acid sequence shown in SEQ ID NO:3 or 9, and/or comprising SEQ ID NO:4
  • the light chain CDR1 of the amino acid sequence, and/or the light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and/or the light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10.
  • the antibody that recognizes NKG2A includes: a heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO: 1, 7, 12, 14 or 16, and a heavy chain CDR2 comprising any amino acid sequence shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and a heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO: 3 or 9, and/or a light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 4, And light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10.
  • the antibody recognizing NKG2A comprises: heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO: 1, 7, 12, 14 or 16, and heavy chain CDR2 comprising any amino acid sequence shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO: 3 or 9, and light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 4, and Light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10.
  • the antibody that recognizes NKG2A includes: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6; or HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10; or HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:3; LCDR3 shown in Q ID NO:6; or comprising HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13 and HCDR3 shown in SEQ ID NO:9;
  • the present invention provides an antibody recognizing NKG2A, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or a variant of the above sequence.
  • the present invention provides an antibody recognizing NKG2A, comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence.
  • heavy and light chain variable region sequences can bind NKG2A
  • heavy and light chain variable region sequences can be "mixed and matched" to generate anti-NKG2A binding molecules of the invention.
  • the invention provides variants of antibodies or fragments thereof that bind NKG2A.
  • the invention thus provides antibodies or fragments thereof having heavy and/or light chain variable regions that are at least 80% identical to the variable region sequences of the heavy or light chains.
  • the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98%, most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99% and 100%.
  • Variants can be obtained by using the antibody described in the present application as the parent antibody, through yeast library screening, phage library screening, point mutation and other methods.
  • antibody A1 and A2 were used as parental antibodies, and the method of phage library screening was used for mutation transformation.
  • the present invention provides antibodies that recognize the same epitope as the aforementioned anti-NKG2A antibodies.
  • the present invention provides antibodies that compete for binding to NKG2A with the aforementioned anti-NKG2A antibodies.
  • the invention provides an antibody that specifically binds NKG2A, said antibody being a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or IgG4 antibody.
  • the antibodies described above do not significantly bind NKG2C, NKG2E or a combination thereof.
  • the antibody binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94 or a combination thereof; and/or,
  • the antibodies bind to cells expressing NKG2A/CD94 and do not significantly bind to cells expressing NKG2C/CD94, NKG2E/CD94, or combinations thereof.
  • Anti-NKG2A antibodies provided herein can be identified, screened for or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. Includes, for example, ELISA, biacore, Western blot and flow cytometry analysis. Suitable assays are described in detail in the Examples.
  • affinity refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the determination of antibody affinity using Biacore as described herein.
  • the "affinity" of an antibody for NKG2A/CD94 is expressed herein as the KD of the antibody.
  • the KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antibody for binding its antigen, the weaker its binding affinity for that particular antigen.
  • EC50 half maximum effect concentration (concentration for 50% of maximal effect, EC50) refers to the concentration that can cause 50% of the maximum effect.
  • antigen refers to a substance recognized and specifically bound by an antigen-binding unit.
  • Antigens can include peptides, proteins, sugars Proteins, polysaccharides and lipids, fractions and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • epitope refers to an antigen or part of an antigen that can be recognized by an antibody, B cell, T cell or engineered cell.
  • an epitope can be a tumor epitope or a pathogen epitope recognized by an antibody; an antibody recognizes multiple epitopes within an antigen. Epitopes can also be mutated.
  • antigenic epitope also known as “antigenic epitope” or “epitope” or “antigenic determinant” includes any determinant or region capable of being bound by an antibody.
  • An epitope is a region of an antigen that is bound by an antibody targeting the antigen, comprising specific amino acids that make direct contact with the antibody.
  • the antigenic epitope may consist of a continuous sequence of NKG2A protein sequence, or may consist of a discontinuous three-dimensional structure of NKG2A protein sequence.
  • the antigens used herein are NAG2A/CD94 heterodimer, NAG2C/CD94 heterodimer, NAG2E/CD94 heterodimer formed by NAG2A extracellular region, NAG2C extracellular region, NAG2E extracellular region and CD94 extracellular region respectively.
  • the present invention also provides immunoconjugates, which include the antibodies described herein, and functional molecules linked thereto.
  • the antibody and the functional molecule can form a conjugate through covalent connection, coupling, attachment, cross-linking and the like.
  • the terms “linked” or “fused” are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or modules by any means including chemical conjugation or recombinant methods.
  • “In-frame fusion” refers to the joining of two or more ORFs to form a contiguous longer ORF in a manner that maintains the correct reading frame of the original open reading frame (ORF).
  • ORF open reading frame
  • the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (the segments are not normally so linked in nature).
  • the reading frames are thus contiguous throughout the fusion fragments, the fragments may be separated physically or spatially by, for example, in-frame linking sequences (eg, "flexons").
  • Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments from different sources or corresponding cDNAs of proteins by genetic recombination technology, including extracellular domains, transmembrane domains and intracellular domains.
  • Chimeric receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor, T Cell Antigen Coupler (TAC).
  • CAR Chimeric Antigen Receptor
  • TAC T Cell Antigen Coupler
  • chimeric antigen receptor includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling junction. domain.
  • the intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a co-stimulatory molecule.
  • the stimulatory molecule is a delta chain bound to a T cell receptor complex; in one aspect, the cytoplasmic signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB (ie, CD137), CD27 and/or CD28.
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides constituting TCR, which can bind to surface antigens on target cells and interact with other polypeptides of the complete TCR complex, usually colocalized on the surface of T cells.
  • the chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the stimulation domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit is operatively linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , and the chimeric T cell receptor is integrated into T cells for expression TCR.
  • T cell antigen coupler T cell antigen coupler, TAC
  • TAC T cell antigen coupler
  • TAC T cell antigen coupler
  • Antigen binding domain including single-chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting groups
  • Extracellular domain single-chain antibody that binds to CD3, so that TAC receptor and TCR receptor are close
  • Transmembrane domain and intracellular domain of CD4 co-receptor Among them, the intracellular domain-linked protein kinase LCK catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell to regulate the activity of the cell via defined signaling pathways by producing secondary messengers or by acting as effectors in response to such messengers.
  • the intracellular signaling domain may include the entire intracellular portion of the molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.
  • the term "primary signaling domain” regulates the initial activation of the TCR complex in a stimulatory manner.
  • the primary signaling domain is elicited by, for example, the binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.).
  • Primary signaling domains acting in a stimulatory manner may comprise immunoreceptor tyrosine activation motifs or signaling motifs of ITAMs.
  • ITAM-containing primary signaling domains examples include, but are not limited to, sequences derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d. primary signal domain.
  • co-stimulatory signal domain refers to "co-stimulatory molecule”, which refers to a signal that combines with a cell-stimulatory signal molecule, such as TCR/CD3, to result in T cell proliferation and/or up-regulation or down-regulation of key molecules. is a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino acid (ND terminus) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during the cellular processing and localization of the CAR to the cell membrane.
  • CD3 delta also known as CD3 Zeta
  • CD3 delta domain is defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of ⁇ comprises residues 52 to 164 of GenBan Accession No. BAG36664.1, a functional ortholog thereof - equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like.
  • the chimeric receptors of the invention are chimeric antigen receptors.
  • the present invention provides a chimeric antigen receptor (Chimeric Antigen Receptor, CAR), which comprises the extracellular binding domain, transmembrane domain and intracellular domain described herein.
  • CAR Chimeric Antigen Receptor
  • the extracellular binding domain (or called structural region) of CAR is derived from mouse or humanized or human monoclonal antibody.
  • Chimeric antigen receptors typically comprise an extracellular antigen-binding domain or an antibody.
  • the extracellular antigen binding region can be fully human.
  • the extracellular antigen binding domain can be humanized.
  • the extracellular antigen binding region may be of murine origin, or a chimera in said extracellular antigen binding region may consist of amino acid sequences from at least two different animals.
  • the extracellular antigen binding region may be non-human.
  • Chimeric antigen receptors can be designed with a variety of antigen-binding domains, including single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding domains (Fab) selected from libraries, single-domain fragments, or natural ligands that engage their cognate receptors.
  • the extracellular antigen binding region may comprise scFv, Fab or natural ligand, and any derivatives thereof.
  • An extracellular antigen binding region can refer to a molecule other than an intact antibody, which can comprise a portion of an intact antibody and can bind to the antigen to which the intact antibody binds.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies, linear antibodies; single chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • An extracellular antigen-binding region such as a scFv, Fab, or natural ligand, can be part of a CAR that determines antigen specificity.
  • the extracellular antigen binding domain can bind any complementary target.
  • Extracellular antigen binding regions can be derived from antibodies of known variable region sequences.
  • Extracellular antigen binding regions can be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, extracellular antigen binding can be obtained from total excision sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TILs). district.
  • TILs tumor infiltrating lymphocytes
  • the binding specificity of the extracellular antigen-binding region of the CAR can be determined by complementarity determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity can be determined by the light and heavy chain CDRs.
  • the extracellular antigen binding region of the CAR includes a hinge or spacer.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be considered as part of the CAR used to provide flexibility to the extracellular antigen-binding region.
  • the hinge can be used to detect a CAR on the cell surface of a cell, particularly when antibodies to detect the extracellular antigen binding region are not functional or available.
  • the length of a hinge derived from an immunoglobulin may need to be optimized, depending on where the epitope on the target is targeted by the extracellular antigen binding region.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of the CD8 ⁇ molecule.
  • the CD8 ⁇ hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 coreceptors and MHC molecules.
  • the cysteine and proline residues can affect the performance of the CAR.
  • the hinge can be adjusted depending on the extracellular antigen binding region used. Hinges can be of any length.
  • the transmembrane domain (or structural region) of CAR can anchor the CAR on the plasma membrane of the cell.
  • the native transmembrane portion of CD28 can be used in CAR. In other cases, it is also possible to use the native transmembrane portion of CD8 ⁇ in the CAR.
  • CD8 may be a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • a "CD8 nucleic acid molecule” may be a polynucleotide encoding a CD8 polypeptide.
  • the transmembrane region may be the natural transmembrane part of CD28, and "CD28” may refer to a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with NCBI reference number: NP_006130 or a fragment thereof having stimulating activity.
  • a "CD28 nucleic acid molecule” may be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion may comprise a CD8 ⁇ region.
  • the (cellular) signaling region of the CAR may be responsible for activating at least one of the effector functions of an immune response cell comprising said CAR.
  • CAR can induce the effector function of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines, such as IL-2, TNF- ⁇ , ⁇ -IFN, etc.
  • the term intracellular signaling region refers to the portion of a protein that transduces the signal for effector functions and directs the cell to specific functions. While usually the entire intracellular signaling region can be used, in many cases it is not necessary to use the entire chain of signaling domains. In some embodiments, truncated portions of intracellular signaling regions are used. In some embodiments, the term intracellular signaling region is thus intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
  • a preferred example of the signaling domain (or called structural region) used in CAR may include the cytoplasmic sequence of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction after target-receptor binding, and any derivative or variant sequences thereof and any synthetic sequences of these sequences with the same functionality.
  • TCR T cell receptor
  • the intracellular signaling region of the CAR may contain a known signaling motif of an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs containing cytoplasmic signaling sequences include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signaling domain is derived from the CD3 delta chain.
  • CD3 delta domain also known as the T cell receptor CD3 delta chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition of several intracellular signal transduction pathways with activation of main effectors of T cells.
  • CD3delta primarily refers to human CD3delta and its isoforms, as known from Swissprot entry P20963, including proteins with substantially identical sequences.
  • the full T cell receptor CD3 delta chain is not required and any derivative thereof comprising the signaling domain of the T cell receptor CD3 delta chain is suitable, including any functional equivalents thereof.
  • the intracellular signal transduction domain (or called structural region) can be selected from any costimulatory domain in Table 1.
  • a domain can be modified such that the identity to a reference domain can be from about 50% to about 100%.
  • Any one of the domains of Table 1 can be modified such that the modified form can comprise about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% identity.
  • the intracellular signaling region of the CAR may further comprise one or more co-stimulatory domains.
  • the intracellular signaling domain may comprise a single co-stimulatory domain, such as the delta chain (first generation CAR) or its combination with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling domain may comprise two co-stimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • CAR proximal signaling proteins associated with CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB/OX40 (TNF-receptor-associated factor adapter protein) pathways, as well as MAPK and Akt activation.
  • CD28 phosphatidylinositol-4,5-bisphosphate 3-kinase
  • 4-1BB/OX40 TNF-receptor-associated factor adapter protein
  • signals generated by CARs may be combined with auxiliary or co-stimulatory signals.
  • chimeric antigen receptor-like complexes can be designed to contain several possible co-stimulatory signaling domains. It is well known in the art that in naive T cells, binding of the T cell receptor alone is not sufficient to induce full activation of the T cell into a cytotoxic T cell. A second co-stimulatory signal is required for full productive T cell activation.
  • T cell activation Several receptors have been reported to provide co-stimulation to T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4-1BB.
  • the signaling pathways used by these co-stimulatory molecules all work synergistically with primary T cell receptor activation signals.
  • the signals provided by these co-stimulatory signaling domains can cooperate with main effector activation signals derived from one or more ITAM motifs (such as CD3zeta signaling domains) and can fulfill the requirements of T cell activation.
  • adding costimulatory domains to chimeric antigen receptor-like complexes can enhance the efficacy and durability of engineered cells.
  • the T cell signaling domain and the co-stimulatory domain are fused to each other to constitute the signaling domain.
  • the present invention also provides various chimeric antigen receptors (CARs) comprising the antibodies or fragments thereof of the present invention, and the CAR-T cells exhibit anti-tumor properties.
  • CARs chimeric antigen receptors
  • cells eg, T cells
  • the viral vector is a lentiviral vector.
  • the cells can stably express the CAR.
  • the NKG2A binding portion of the CAR is scFv, which maintains equivalent affinity and binding capacity compared with the Fab antibody from which it is derived, for example, it binds the same antigen with comparable efficacy.
  • the antibody fragment is functional in that it provides a biochemical response, such as activation of an immune response, inhibition of signaling initiation from its target antigen, inhibition of kinase activity, and the like.
  • the anti-NKG2A antigen-binding domain of the CAR is a fully human antibody fragment.
  • the CAR of the present invention combines the antigen-binding domain of a specific antibody with an intracellular signaling molecule.
  • intracellular signaling molecules include, but are not limited to, CD3delta, 4-1BB, and CD28 signaling modules and combinations thereof.
  • the NKG2A-CAR comprises at least one intracellular signaling domain selected from the group consisting of CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3 ⁇ signaling domain, and any combination thereof. In one aspect, the NKG2A-CAR comprises at least one intracellular signaling domain derived from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.
  • sequence of NKG2A-CAR can be:
  • a chimeric antigen receptor having an extracellular domain set forth in SEQ ID NO:64, a hinge domain set forth in SEQ ID NO:95, a transmembrane domain set forth in SEQ ID NO:97, a co-stimulatory signaling domain set forth in SEQ ID NO:101, and a primary signaling domain (A4-BBZ) set forth in SEQ ID NO:105; or
  • Chimeric antigen receptor two has an extracellular domain shown in SEQ ID NO:66, a hinge domain shown in SEQ ID NO:95, a transmembrane domain shown in SEQ ID NO:97, a co-stimulatory signal domain shown in SEQ ID NO:101, and a primary signal domain (A5-BBZ) shown in SEQ ID NO:105.
  • amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 115 or 116.
  • transmembrane domain and intracellular domain of the chimeric antigen receptor above can be replaced by conventional transmembrane domains and intracellular domains by those skilled in the art, and all of them fall within the protection scope of the present application.
  • nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribose nucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
  • sequence when used to refer to a nucleotide sequence includes DNA or RNA, and may be single- or double-stranded.
  • target sequence refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of a CRISPR complex.
  • a target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • sequence identity determines the percent identity by comparing two best-matched sequences over a comparison window (e.g., at least 20 positions), wherein portions of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions (i.e., gaps), e.g., a gap of 20% or less (e.g., 5 to 15%, or 10 to 12%) for the two best-matched sequences compared to a reference sequence (which does not contain additions or deletions).
  • a comparison window e.g., at least 20 positions
  • portions of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions (i.e., gaps), e.g., a gap of 20% or less (e.g., 5 to 15%, or 10 to 12%) for the two best-matched sequences compared to a reference sequence (which does not contain additions or deletions).
  • Percentages are typically calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to yield the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity.
  • transfection refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising a An expression control sequence operably linked to the sequence.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system.
  • Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • vector is a composition comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • Non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, and the like.
  • lentivirus refers to a genus of the Retroviridae family. Retroviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide the means to achieve significant levels of gene transfer in vivo.
  • endogenous means that a nucleic acid molecule or polypeptide etc. is derived from the organism itself.
  • exogenous refers to a function of a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not expressed endogenously in the organism itself, or the expression level is insufficient to achieve overexpression.
  • exogenous protein used herein may be a protein introduced into cells exogenously that recognizes a target antigen, such as an exogenous receptor (ie, the aforementioned “chimeric receptor” herein).
  • host refers to a recipient receiving a transplant, and in some embodiments, may be an individual, such as a human, receiving exogenous cells implanted.
  • isolated means separated from cellular components or other components in which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are normally associated in their natural state.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not need to be “isolated” to distinguish it from its naturally occurring counterpart.
  • a “concentrated”, “isolated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is distinguishable from its naturally occurring counterpart because the concentration or number of molecules per volume is greater (“concentrated") or less than ("diluted”) the concentration of its naturally occurring counterpart.
  • the degree of enrichment can be measured on an absolute basis, such as weight per solution volume, or can be measured relative to another potential interferent present in the source mixture.
  • the preferred enrichment degree of the technical solution of the present invention is higher.
  • 2-fold enrichment is preferred
  • 10-fold enrichment is more preferred
  • 100-fold enrichment is more preferred
  • 1000-fold enrichment is more preferred.
  • An "isolated" substance can also be provided by artificial means of assembly, for example, by chemical synthesis or recombinant expression.
  • the present invention provides an isolated nucleic acid encoding an NKG2A-recognizing antibody or a fragment thereof, a vector, and a host cell comprising the nucleic acid or vector.
  • Nucleic acids can be located in intact cells, in cell lysates, or in partially or substantially purified form.
  • Nucleic acids of the invention can be obtained using standard molecular biology techniques, for example, cDNAs encoding antibody light and heavy chains or encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • cDNAs encoding antibody light and heavy chains or encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from an immunoglobulin gene library eg, using phage display technology
  • one or more nucleic acids encoding the antibody can be recovered from the library.
  • Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary with the host cell used. change.
  • the nucleic acid molecule of the present invention is selected from SEQ ID NO: 19, 23, 27, 29, 31 or 33 encoding the variable region of the heavy chain, and/or selected from SEQ ID NO: 21 or 25 encoding the variable region of the light chain.
  • it is a nucleic acid molecule comprising a heavy chain variable region sequence of SEQ ID NO: 19, and a light chain variable region sequence comprising SEQ ID NO: 21; or a heavy chain variable region sequence comprising SEQ ID NO: 23, and a light chain variable region sequence comprising SEQ ID NO: 25; or a heavy chain variable region sequence comprising SEQ ID NO: 27, and a light chain variable region sequence comprising SEQ ID NO: 21; or A heavy chain variable region sequence comprising SEQ ID NO:29, and a light chain variable region sequence comprising SEQ ID NO:25; or a heavy chain variable region sequence comprising SEQ ID NO:31, and a light chain variable region sequence comprising SEQ ID NO:25; or a heavy chain variable region sequence comprising SEQ ID NO:33, and a light chain variable region sequence comprising SEQ ID NO:25.
  • one or more vectors comprising the nucleic acids described above are provided.
  • cell refers to a cell of human or non-human animal origin.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include transformed primary cells and progeny derived therefrom (regardless of the number of passages).
  • the nucleic acid content of the progeny may not be identical to that of the parental cells and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • NKG2A or NKG2A/CD94 positive host cell refers to a host cell expressing NKG2A/CD94 on the cell surface, which can be detected, for example, by flow cytometry using antibodies that specifically recognize a combined epitope on CD94 and NKG2A or an epitope on NKG2A alone.
  • the host cells are immune effector cells.
  • immune effector cells refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cells may be autologous T cells, allogeneic T cells, allogeneic T cells.
  • the NK cells may be allogeneic NK cells.
  • immune effector function or immune effector response refers to immune effector cells, eg, functions or responses that enhance or facilitate immune attack of target cells.
  • immune effector function or response refers to the properties of T cells or NK cells that promote killing of target cells or inhibit growth or proliferation.
  • artificially engineered cell with immune effector cell function refers to a cell or cell line that does not have immune effector after being artificially modified or stimulated by a stimulant, the cell acquires immune effector cell function.
  • 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
  • T cells may be pluripotent stem cells derived from bone marrow that differentiate and mature into immunocompetent mature T cells within the thymus.
  • T cells may be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as “T cells” may be cells comprising at least one T cell subset: memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells (Tem).
  • Tscm cells memory stem cell-like T cells
  • Tcm central memory T cells
  • effector T cells Tef, Teff
  • Tregs regulatory T cells
  • effector memory T cells Tem
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood collected from an individual using any number of techniques known to those of skill in the art, such as Ficoll(TM) isolation.
  • the cells from the circulating blood of the individual are obtained by apheresis.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps.
  • T cells can be obtained from a healthy donor, or from cells derived from a patient diagnosed with a tumor.
  • peripheral blood mononuclear cell refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes and the like.
  • activation and “activation” are used interchangeably and can refer to the process by which a cell transitions from a quiescent state to an active state.
  • the process can include responses to antigenic, phenotypic or genetic changes in migration and/or functional activity status.
  • activation may refer to the process of gradual activation of NK cells and T cells.
  • T cell activation or “T cell activation” refers to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • a host cell comprising the nucleic acid described above.
  • the host cell comprises (e.g., is transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, eg, Chinese Hamster Ovary (CHO) cells or lymphocytes (eg, YO, NSO, Sp20 cells).
  • the host cell expresses a chimeric receptor of the invention.
  • the host cells comprise T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, and/or stem cell-derived immune effector cells.
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells; preferably, the T cells are autologous/allogeneic T cells; preferably, the T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), ⁇ T cells or combinations thereof.
  • the host cell binds to cells expressing NKG2A/CD94 and does not significantly bind NKG2C/CD94, NKG2E/CD94, or a combination thereof.
  • the host cell also carries coding sequences for exogenous cytokines.
  • the host cell may also express another chimeric antigen receptor in addition to the antigen-binding receptors described above.
  • the host cell may also express chemokine receptors.
  • the host cell can also express a safety switch.
  • the host cell is capable of killing activated NK cells.
  • a method of making an anti-NKG2A antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • the nucleic acid encoding the antibody of the present invention can be incorporated into an expression vector.
  • expression vectors are available for protein expression.
  • Expression vectors may include self-replicating extrachromosomal vectors, or vectors that integrate into the host genome.
  • Expression vectors useful in the present invention include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems.
  • a variety of expression vectors are available commercially or otherwise, as known in the art.
  • Antibodies may be expressed in the present invention.
  • the host cell is administered in combination with a drug that enhances its function, preferably, a chemotherapy drug; and/or the host cell is administered in combination with a drug that improves one or more side effects associated therewith; and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting other than NKG2A.
  • endogenous TCR, B2M, NKG2A and/or CIITA in cells are knocked out using a CRISPR system comprising gRNA provided by the present invention.
  • Genetic modification of cells eg, T cells or NKT cells
  • T cells or NKT cells can be accomplished by transducing a substantially homogeneous population of cells with a recombinant nucleic acid molecule.
  • TCR, B2M, NKG2A and/or CIITA genes in the cells are knocked out, TCR, B2M, NKG2A and/or HLA-II are expressed low or not in the cells.
  • Low or no expression of TCR, B2M, NKG2A and/or HLA-II means that the expression of CR, B2M, NKG2A and/or HLA-II in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • low or no expression of CR, B2M, NKG2A and/or HLA-II means that the content of CR, B2M, NKG2A and/or HLA-II in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.
  • the expression or content of proteins in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies to CR, B2M, NKG2A and/or HLA-II.
  • the donor due to the immunogenetic differences between the donor and the recipient (or host), during exogenous donor transplantation, the donor as an exogenous graft will be recognized and attacked by immune cells (such as NK cells) in the host, and then inhibit or eliminate the donor, resulting in host-versus-graft reaction (HVGR).
  • immune cells such as NK cells
  • HVGR host-versus-graft reaction
  • the present invention provides immune cells with low or no expression of endogenous HLA-II/B2M.
  • GVHD graft-versus-host disease
  • the donor T lymphocytes will recognize the antigens on the normal tissues of the host, amplify and release a series of cytokines, which greatly enhance the immune response of the graft to the host antigens and attack the host cells.
  • the present invention provides immune cells with low or no expression of endogenous HLA-II/TCR.
  • the present invention uses the CRISPR system to knock out the gene TRAC of the alpha chain of the endogenous TCR to prepare cells with low or no expression of the endogenous TCR.
  • the expression of endogenous NKG2A in donor immune cells of exogenous grafts is up-regulated, and will be killed by immune cells that recognize NKG2A in the composition of the present invention.
  • low expression or no expression of NKG2A may release the inhibitory effect of immune cells themselves, thus exerting stronger anti-tumor ability.
  • the present invention provides immune cells with low or no expression of endogenous HLA-II/NKG2A.
  • the above-mentioned immune cells did not significantly activate allogeneic immune cells.
  • the above-mentioned immune cells can reduce the allogeneic immune rejection.
  • the above-mentioned immune cells that recognize tumor antigens and/or immune cells that recognize NKG2A polypeptides and tumor antigens can significantly kill tumor cells without significantly activating allogeneic immune cells.
  • the above-mentioned immune cells that recognize tumor antigens and/or immune cells that recognize NKG2A polypeptides and tumor antigens can significantly kill tumor cells with low allogeneic immune rejection.
  • the antibodies, immune conjugates containing the antibodies, chimeric receptors, and host cells of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents.
  • the composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • the composition includes another therapeutic agent.
  • the other therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof.
  • such therapeutic agents include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and norvibin(TM) (vinorelbine, 5'-dehydrogensulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL), and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, Examples include etoposide, teniposide, and midoxezoz; alkylating agents cisplatin, cyclophosphamide, mechlorethamine, trimethylenethiophosphoramide, carmustine, busul
  • mitotic inhibitors vinca alkal
  • the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil.
  • additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides), and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2, among others.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides), and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid, and tissue inhibitors of
  • sugars such as lactose, glucose and Sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.
  • sugars such as lactose, glucose and Sucrose
  • starches such as cornstarch and potato starch
  • cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose
  • tragacanth powder mal
  • compositions described herein may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, eg, Berge, S.M et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, and those derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like.
  • Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
  • antioxidants include, but are not limited to: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, alcohol Petroleum acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • composition of the present invention can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dosage beneficial to the patient.
  • the mode of administration can be, for example, parenteral administration (such as injection) or other therapeutic modes.
  • parenteral administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • compositions may be isotonic, ie, they may have the same osmotic pressure as blood and tear fluid.
  • the desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickener, if desired.
  • Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.
  • the preferred concentration of thickener will depend on the agent chosen. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, eg a liquid dosage form.
  • kits comprising the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein.
  • a kit may comprise an effective amount of a compound described herein comprising one or more unit dosage forms.
  • Therapeutic or prophylactic compositions of antibodies, chimeric receptors, nucleic acids or host cells comprise kits comprise sterile containers that may contain therapeutic or prophylactic compositions; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, blister packs, or other suitable container forms known in the art.
  • Such containers may be made of plastic, glass, laminated paper, foil, or other materials suitable for holding medications.
  • the kit comprises an antibody, immune conjugate, chimeric receptor, nucleic acid or host cell described herein, and instructions for administering the antibody, immune conjugate, chimeric receptor, nucleic acid or host cell described herein to an individual.
  • the instructions generally include methods of treating or preventing cancer or tumors using the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein.
  • the kit comprises a host cell described herein, and can include from about 1 x 104 cells to about 1 x 106 cells. ⁇ , ⁇ 1 ⁇ 10 5 ⁇ , ⁇ 1 ⁇ 10 6 ⁇ , ⁇ 1 ⁇ 10 7 ⁇ , ⁇ 4 ⁇ 10 7 ⁇ , ⁇ 5 ⁇ 10 7 ⁇ , ⁇ 6 ⁇ 10 7 ⁇ , ⁇ 6 ⁇ 10 7 ⁇ ,8 ⁇ 10 7 ⁇ , ⁇ 9 ⁇ 10 7 ⁇ , ⁇ 1 ⁇ 10 8 ⁇ , ⁇ 2 ⁇ 10 8 ⁇ , ⁇ 3 ⁇ 10 8 ⁇ , ⁇ 4 ⁇ 10 8 ⁇ , ⁇ 5 ⁇ 10 8 ⁇ , ⁇ 6 ⁇ 10 8 ⁇ , ⁇ 6 ⁇ 10 8 ⁇ , ⁇ 8 ⁇ 10 8 ⁇ , ⁇ 9 ⁇ 10 8 ⁇ , ⁇ 1 ⁇ 10 9 ⁇ , ⁇ 2 ⁇ 10 9 ⁇ , ⁇ 3 ⁇ 10 9 ⁇ , ⁇ 4 ⁇ 10 9 ⁇ , ⁇ 5 ⁇ 10 9 ⁇ , ⁇ 6 ⁇ 10 9 ⁇ , ⁇ 8 ⁇ 10 9 ⁇ , ⁇ 9 ⁇ 10 9 ⁇ , ⁇ 1 ⁇ 10 10 ⁇ , ⁇ 2 ⁇ 10 9
  • the kit can include allogeneic cells.
  • a kit can include a cell that can contain a genomic modification.
  • a kit may comprise "ready-to-use" cells.
  • a kit can include cells that can be expanded for clinical use. In some cases, kits may contain contents intended for research purposes.
  • the instructions include at least one of: a description of the therapeutic agent; dosage regimen and administration for treating or preventing a tumor or symptoms thereof; precautions, warnings, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and/or citations. Instructions may be printed directly on the container (if present), or as a label on the container, or as a separate sheet, booklet, card or folder provided within or in the container. In some embodiments, the instructions provide a method of administering an antibody described herein for treating or preventing a tumor. In certain instances, the instructions provide for administering an antibody of the invention before, after, or concurrently with administration of a chemotherapeutic agent.
  • module refers to positive or negative changes. Examples of adjustments include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes. In a specific embodiment, refers to a negative change.
  • treatment refers to interventions in an attempt to modify the disease process, either prophylaxis or intervention in the clinicopathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing down the progression of the disease, improving or relieving the disease, remission or improving the prognosis, etc.
  • prevention refers to interventions that are attempted in advance of a disease such as rejection of a cell transplant.
  • tumor antigen refers to an antigen emerging or overexpressed during the onset, progression of a hyperproliferative disease.
  • a hyperproliferative disorder of the invention refers to a tumor.
  • the tumor antigens described in the present invention may be solid tumor antigens or blood tumor antigens.
  • the tumor antigens of the present invention include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; B7H6; KIT (CD117); Interleukin 13 receptor subunit alpha (IL-13R ⁇ ); Interleukin 11 receptor alpha (IL-11R ⁇ ); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Tyrosinase; Mesothelin; ); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR- ⁇ ); IX (CAIX); LMP
  • Pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; virus antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
  • Subjects of the invention refers to any animal, such as a mammal or a marsupial.
  • Subjects of the invention include, but are not limited to, humans, non-human primates (such as rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • the term "effective amount” refers to an amount that provides a therapeutic or prophylactic benefit.
  • anti-NKG2A antibodies Any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein can be used in methods of treatment.
  • any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit for use as a medicament is provided.
  • any anti-NKG2A antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in treating a disease is provided.
  • any anti-NKG2A antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in a method of treatment is provided.
  • the invention provides any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified host cell, pharmaceutical composition or kit for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual an effective amount of any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified immune host cell, pharmaceutical composition or kit. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the present invention provides the use of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit in the preparation or formulation of a medicament.
  • the medicament is used to treat a disease.
  • the medicament is for use in a method of treating a disease comprising administering an effective amount of the medicament to a diseased individual.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the invention provides methods for treating a disease.
  • the method comprises administering to an individual having an HLA-E expressing disease an effective amount of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the invention provides a pharmaceutical formulation comprising any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein, eg, for use in any of the aforementioned methods of treatment.
  • the pharmaceutical formulation comprises any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits and pharmaceutically acceptable carriers provided herein.
  • the pharmaceutical formulation comprises any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein and at least one additional therapeutic agent.
  • the pharmaceutical formulation is used to treat a disease.
  • the pharmaceutical formulation is administered to a diseased individual.
  • An "individual" according to any of the above embodiments is preferably a human.
  • the present invention provides a method for the preparation of a medicament or a pharmaceutical preparation, the method comprising mixing any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the aforementioned methods of treatment.
  • the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.
  • any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits of the invention can be used in therapy alone or in combination with other agents.
  • any of the anti-NKG2A antibodies, immunoconjugates, chimeric antigen receptor modified host cells, pharmaceutical compositions or kits of the invention can be co-administered with at least one additional therapeutic agent.
  • Such combination therapy as described above includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the administration of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit of the invention may occur before, simultaneously with, and/or after the administration of the additional therapeutic agent or agent.
  • administration of any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor-modified host cell, pharmaceutical composition or kit of the invention and administration of the additional therapeutic agent occur within about one month, or within about one, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days of each other.
  • any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits of the invention can be administered by any suitable means, including parenteral, intrapulmonary or intranasal administration, and, if therapeutically warranted, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic.
  • a variety of dosing regimens are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • a formulation comprising a population of immunoreactive cells administered to an individual comprises a plurality of immunoreactive cells effective to treat and/or prevent a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • a formulation comprising about 1 x 104 to about 1 x 1010 immunoreactive cells is administered.
  • the formulation will contain about 1 x 105 to about 1 x 109 immunoreactive cells, about 5 x 105 to about 5 x 108 immunoreactive cells, or about 1 x 106 to about 1 x 107 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to an individual will vary between wide ranges. Your doctor will ultimately determine the proper dosage to use.
  • chimeric receptors are used to stimulate host cell-mediated immune responses.
  • a T cell-mediated immune response is an immune response that involves the activation of T cells.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells displaying foreign antigen epitopes on their surface, such as cancer cells displaying tumor antigens.
  • chimeric antigen receptors are used to provide anti-tumor immunity in mammals. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.
  • methods of treating a subject with a tumor may involve administering to the subject in need of treatment one or more host cells described herein.
  • the host cells can bind tumor target molecules and induce cancer cell death.
  • the present invention also provides a method of treating a pathogenic infection in an individual comprising administering to said individual a therapeutically effective amount of a host cell of the invention.
  • the frequency of administration of the host cells of the invention will depend on factors including the disease being treated, elements of the particular host cell and the mode of administration. For example, it can be administered 4 times a day, 3 times, 2 times a day, or once a day, every other day, every three days, every four days, every five days, once every six days, once a week, once every eight days, once every nine days, every ten days, once a week, or twice a month. As described herein, due to the improved viability of the host cells of the present application, they can not only be administered in a lower therapeutically effective amount than similar host cells that do not express exogenous type I interferon, but also can be administered at a lower frequency to obtain at least similar and preferably more significant curative effects.
  • the present invention provides an antibody specifically binding to NKG2A, which is a fully human antibody with low immunogenicity and few possible clinical side effects;
  • the antibody of the present invention can effectively block the combination of HLA-E of tumor cells and NKG2A/CD94 of NK cells, reduce the inhibitory effect of tumor cells expressing HLA-E on NK cells through the NKG2A/CD94 pathway, enhance the killing effect of NK cells on tumor cells, and show good anti-tumor effects.
  • T cells expressing NKG2A-CAR prepared by the antibody of the present invention can kill NK cells; T cells prepared by using the antibody of the present invention targeting both tumors and NK cells can kill NK cells and enhance their anti-tumor effect; UCAR-T cells expressing NKG2A-CAR prepared by the antibody of the present invention can resist the killing of NK cells, enhance their survival ability, and can cooperate with the anti-tumor effect of tumor-targeting T cells or CAR-T cells.
  • the eukaryotic expression plasmid V152S was used to construct vectors expressing NKG2A/CD94 and NKG2C/CD94 heterodimers respectively.
  • ⁇ mFc( ⁇ SEQ ID NO:84 ⁇ ) ⁇ NKG2C ⁇ ( ⁇ 73 ⁇ ) ⁇ (G 4 S) 3 ( ⁇ SEQ ID NO:110 ⁇ ) ⁇ CD94 ⁇ ( ⁇ SEQ ID NO:77 ⁇ ) ⁇ mFc-NKG2C-CD94 ⁇ V152S, ⁇ V152S-mFc-NKG2C-CD94( ⁇ 1) ⁇
  • V152S-mFc-NKG2A-CD94 and V152S-mFc-NKG2C-CD94 were respectively transfected into HEK293 cells (China Center for Type Culture Collection (CCTCC)) and cultured for 7 days.
  • the culture medium was centrifuged to obtain the supernatant, and the Mabselect Sure column was used for affinity purification to obtain antigen NKG2A/CD94 and NKG2C/CD94 heterodimers, respectively.
  • the phage display library used in the present invention is a phage library constructed by our company with a capacity of 1E+11.
  • a highly specific Fab fragment for NKG2A/CD94 heterodimer was obtained using screening methods known to those skilled in the art.
  • the supernatant was taken and added to an immunotube coated with mFc-NKG2A-CD94 for 1.5 hours, then the non-specific phages were washed away, the bound phages were eluted and infected with Escherichia coli TG1 in logarithmic growth phase.
  • the eluted phages were expanded and purified by PEG/NaCl precipitation for the next round of screening. A total of three rounds of screening were performed to enrich the Fab phage clones specifically binding to the NKG2A/CD94 heterodimer. Positive clones were identified by standard ELISA methods against NKG2A.
  • Antibody specificity was verified by ELISA using mFc-NKG2C-CD94 as an irrelevant antigen. A total of 1504 clones were screened, among which 50 clones only bound to mFc-NKG2A-CD94 and did not bind to mFc-NKG2C-CD94. After sequencing, 2 clones were obtained. These two clones were expressed and purified to obtain antibodies A1 and A2 in Fab form.
  • the amino acid sequence of HCDR1 of A1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6.
  • the amino acid sequence of the heavy chain variable region of A1 is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20.
  • the amino acid sequence of the heavy chain of A1 is shown in SEQ ID NO:34, and the amino acid sequence of the light chain is shown in SEQ ID NO:42.
  • the amino acid sequence of HCDR1 of A2 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR1 is shown in SEQ ID As shown in NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:10.
  • the amino acid sequence of the heavy chain variable region of A2 is shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24.
  • the amino acid sequence of the heavy chain of A2 is shown in SEQ ID NO:35, and the amino acid sequence of the light chain is shown in SEQ ID NO:46.
  • A1-IgG4 Simultaneous construction and eukaryotic expression of purified IgG4 antibodies resulted in two antibodies specifically binding to NKG2A/CD94 heterodimers, named A1-IgG4 and A2-IgG4.
  • the heavy chain amino acid sequence of A1-IgG4 is shown in SEQ ID NO:40, and the light chain amino acid sequence is shown in SEQ ID NO:42.
  • the heavy chain amino acid sequence of A2-IgG4 is shown in SEQ ID NO:44, and the light chain amino acid sequence is shown in SEQ ID NO:46.
  • Antigens mFc-NKG2A-CD94 and mFc-NKG2C-CD94 were diluted with PBS to a concentration of 5 ⁇ g/ml, and coated overnight at 4°C.
  • 2% MPBS silkmed milk powder/PBS
  • primary antibody A1 (10 ⁇ g/ml) or A2 (10 ⁇ g/ml) was added, and incubated for 1 hour at room temperature; after washing with PBST for 5 times, secondary antibody (anti-Flag-HRP, 1:4000, Sigma) was added to continue incubation at room temperature for 1 hour; after washing with PBST for 5 times, TMB was developed and the OD450 value was read with a microplate reader.
  • NA is the blank control.
  • Antibodies A1 and A2 both specifically bind to NKG2A/CD94 heterodimer, but not to NKG2C/CD94 heterodimer.
  • the mFc-NKG2A-CD94 prepared above was diluted with PBS to a concentration of 2.5 ⁇ g/ml, and coated overnight at 4°C.
  • Chip CM5 (ID: 180925-0245: 1738291) was coated with Anti-huFc antibody, A1-IgG4 and A2-IgG4 antibodies were used as ligands, and mFc-NKG2A-CD94 was used as mobile phase (the curves in Figure 4 represent concentrations of 150nM, 50nM, 16.67nM, 5.56nM and 1. 85nM), the regeneration reagent was 3M MgCl 2 , at 25°C, and the experimental data were processed with Biacore T200Evaluation Software2.0 software. Select "surface bound”, select "local” for Rmax, and use a 1:1 langmuir model for fitting. The results are shown in Figure 4, the affinity KD values of A1-IgG4 and A2-IgG4 binding to NKG2A/CD94 heterodimer were 4.41nM and 4.30nM, respectively.
  • CD94 full-length including CD94 full-length, Flag tag
  • NKG2A-CD94 including NKG2A full-length, F2A, CD94 full-length, Flag tag
  • NKG2C-CD94 including NKG2C full-length, F2A, CD94 full-length, Flag tag
  • NKG2E-CD94 including NKG2 E full length, F2A, CD94 full length, Flag tag
  • the CHO-K1 stable cell line CHOK1-CD94 expressing CD94 the CHO-K1 stable cell line CHOK1-NKG2A-CD94 expressing NKG2A-CD94, the CHO-K1 stable cell line CHOK1-NKG2C-CD94 expressing NKG2C-CD94 and NKG2C-CD94 were successfully obtained.
  • the G2E-CD94 CHO-K1 stably transfected cell line CHOK1-NKG2E-CD94 (see Figure 5 for the vector map), and the positive rates were all greater than 90%.
  • the full-length base sequence of CD94 is SEQ ID No.128, the full-length base sequence of NKG2A is SEQ ID No.69, the full-length base sequence of NKG2C is SEQ ID No.124, the full-length base sequence of NKG2E is SEQ ID No.126, the base sequence of the Flag tag is SEQ ID No.112, and the base sequence of F2A is SEQ ID No.108 .
  • A1 and A2 as parental antibodies, two phage libraries were constructed using conventional biological techniques, one randomized the CDR1 and CDR2 of the light chain, and the other randomized the CDR1 and CDR2 of the heavy chain. Then screen against the antigen, and screen out high-affinity antibodies, that is, variants of A1 or A2, by ELISA technology and the like.
  • a template plasmid was constructed based on antibody A1 (Fab).
  • Fab antibody A1
  • the full-length Fab sequence was digested with NcoI and NotI, ligated into the same digested template plasmid by T4 ligase, and electrotransformed into TG1 competent cells with a storage capacity of 2.6x10 9 .
  • the construction of the antibody A2 affinity maturation library is similar to that of A1, and the template plasmid is constructed based on the antibody A2 (Fab). Using the same primers as A1 to randomize CDR1 and CDR2 of the light chain, the resulting phage library has a capacity of 1.89x10 9 .
  • the first round of screening was coated with the antigen mFc-NKG2A-CD94 at a concentration of 5 ⁇ g/ml and cleared with the antigen mFc-NKG2C-CD94 at a concentration of 5 ⁇ g/ml; the second round of screening was coated with the antigen mFc-NKG2A-CD94 at a concentration of 1 ⁇ g/ml and cleared with the antigen mFc-NKG2E-CD94 at a concentration of 2 ⁇ g/ml.
  • positive clones were determined by ELISA.
  • the amino acid sequences of the heavy chain variable regions of six antibodies A1, A2, A3, A4, A5 and A6 were compared.
  • the heavy chain variable region sequence similarity between A3 and parent A1 was 98.3%.
  • the heavy chain variable region sequence similarity between A4 and the parent A2 was 94.8%.
  • the heavy chain variable region sequence similarity between A5 and the parent A2 was 94.8%.
  • the heavy chain variable region sequence similarity between A6 and the parent A2 was 93.1%.
  • the antigen mFc-NKG2A-CD94 was diluted with PBS to a concentration of 2 ⁇ g/ml, and coated overnight at 4°C.
  • 2% MPBS silkmed milk powder/PBS
  • primary antibodies were added (A3, A4, A5, A6: 25 ⁇ g/mL initial 5-fold gradient dilution and 8 gradients), and incubated at room temperature for 1 hour; after washing with PBST for 5 times, secondary antibody (anti-Flag-HRP, 1:4000, Sigma) was added to continue incubation at room temperature for 1 hour; after washing with PBST for 5 times, TMB was developed and OD was read with a microplate reader 450 values.
  • CHOK1-NKG2A-CD94 cells were counted and spread on a U-shaped bottom plate, with about 2 ⁇ 105 cells per well, and then incubated with primary antibody (A1, A2, A3, A4, A5, A6: 25 ⁇ g/mL initial 5-fold gradient dilution and 8 gradients) and secondary antibody (Anti-Fab-FITC: 1:200, Jackson ImmunoResearch), and then detected the fluorescence intensity with a flow cytometer.
  • the four-parameter fitting was performed with the primary antibody concentration as the abscissa and the calibrated mean fluorescence intensity (MFI) as the ordinate through the GraphPad Prism5 software, and the EC50 value was calculated.
  • MFI mean fluorescence intensity
  • Figure 10 the antibody (Fab format) binds to the overexpressed NKG2A/CD94 heterologous
  • the EC50 values of the dimerized CHO-K1 cells are shown in Table 9, and the binding ability of the antibody cells after affinity maturation is significantly better than that of the corresponding parent antibody.
  • the antigen mFc-NKG2A-CD94 was diluted with PBS to a concentration of 2 ⁇ g/ml, and coated overnight at 4°C.
  • the antibody (IgG4 format) binds to the NKG2A/CD94 heterodimer in a concentration gradient-dependent manner, and the EC50 values are shown in Table 10.
  • the CHOK1-NKG2A-CD94 cells were counted and laid on a U-shaped bottom plate, with about 2 ⁇ 105 cells per well, and then the primary antibody (A1-IgG4, A2-IgG4, A3-IgG4, A4-IgG4, A5-IgG4, A6-IgG4: 25 ⁇ g/mL initial 5-fold serial dilution 8 gradients) and secondary antibody (Anti-Fab-FITC: 1:200 , Jackson ImmunoResearch) incubation, followed by flow cytometer for fluorescence intensity detection.
  • the primary antibody A1-IgG4, A2-IgG4, A3-IgG4, A4-IgG4, A5-IgG4, A6-IgG4: 25 ⁇ g/mL initial 5-fold serial dilution 8 gradients
  • Anti-Fab-FITC 1:200 , Jackson ImmunoResearch
  • the antibodies (IgG4 format) all bind to CHO-K1 cells overexpressing NKG2A/CD94 heterodimers in a concentration gradient-dependent manner.
  • the EC50 values are shown in Table 11, which indicates that the binding activity of the antibodies after affinity maturation is improved.
  • Table 11 The EC50 of antibody (IgG4 form) binding to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer
  • Chip CM5 (ID: 180925-0245: 1738291) was coated with Anti-huFc antibody, A3-IgG4, A4-IgG4, A5-IgG41, A6-IgG4 antibodies were used as ligands, mFc-NKG2A-CD94 was used as mobile phase (the curves in Figure 13 from top to bottom represent concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.70 nM and 1.23 nM), the regeneration reagent is 3M MgCl 2 , 25°C.
  • the experimental data were processed with Biacore T200 Evaluation Software2.0 software. Select "surface bound”, select Rmax "local”, using a 1:1 Langmuir model for fitting.
  • the results are shown in Figure 13.
  • the KD value of the binding affinity between the obtained antibody (IgG4 format) and the NKG2A/CD94 heterodimer is shown in Table 12, and the affinity of the antibody after affinity maturation is about 12-18 times higher than that of the parental antibody.
  • the plasmids pET22b-HLA-E and pET22b- ⁇ 2m comprising the fragment HLA-E-avi- (comprising the extracellular segment of human HLA-E (sequence shown in SEQ ID NO: 79), the avi tag (sequence shown in SEQ ID NO: 114), and fragment ⁇ 2m (sequence shown in SEQ ID NO: 81) were respectively transferred into BL21 strains (see Figure 1 for the vector) by plasmid transduction. 4).
  • Prokaryotic induction of BL21 strain was used to induce expression, and the bacteria were collected and then ultrasonically disrupted to collect inclusion body precipitates.
  • the inclusion body precipitate was washed and then dissolved with 8M urea, further purified by an anion exchange column and detected by electrophoresis, with a purity of over 90%.
  • Add 5 mg of VMA nonapeptide (amino acid sequence as shown in SEQ ID NO: 82, synthesized by Jill Biochemical) to 100 ml of refolding solution, and then add purified HLA-E and ⁇ 2m at a molar ratio of 1:2 to obtain a refolding complex, which is then dialyzed against PBS 5% glycerol.
  • the product was purified by molecular sieves, the target peak was collected, concentrated and replaced to 10mM Tris pH 8.0, and stored at -80°C.
  • the complex was biotin-labeled with birA enzyme, and the labeled product and SA-PE (PE-labeled SA antibody (BD Horizon TM )) were gently mixed at a ratio of 1:4 to obtain the final HLA-E tetramer.
  • NK cells were purified from peripheral blood PBMCs by NK Cell Isolation Kit (Miltenyi Biotec), and treated with NK cells containing 500IU/ml IL-2 and 150IU/ml IL-15 Medium (Miltenyi Biotec) was cultured for 8 days to collect NKG2A positive NK cells (NKG2A + NK), and the positive rate was 86%.
  • the concentration of HLA-E tetramer started from 5 ⁇ g/ml, 5-fold serial dilution, and the concentration of the blank hole was 0. After co-incubating with NK cells at 4°C for 45 min, they were washed 3 times with PBS containing 1% FBS, and then the PE fluorescence signal was detected by flow cytometry. The results are shown in Figure 15.
  • the HLA-E tetramer was significantly combined with the NK cells expressing NKG2A/CD94 in a concentration-gradient-dependent manner, with an EC50 of 0.1029 ⁇ g/ml.
  • Example 4 Take the NKG2A-positive NK cells (NKG2A + NK) prepared in Example 4, add HLA-E-PE and anti-NKG2A antibody at the same time and incubate at 4° C. for 45 minutes to detect the intensity of PE fluorescence signal.
  • the EC50 value of HLA-E-PE concentration was selected, that is, 0.103 ⁇ g/ml, the antibody concentration started from 10 ⁇ g/ml, and was diluted to 0.000128 ⁇ g/ml in a 5-fold gradient, and the blank group only contained HLA-E-PE. After co-incubation, wash 3 times with PBS containing 1% FBS, and then detect PE fluorescence signal by flow cytometer to obtain MFI value.
  • Antibodies (IgG4 form) block the IC50 value of HLA-E binding to NK cells
  • NKG2A antibody can reduce the inhibition of NK cell activity by target cells expressing HLA-E
  • the NKG2A + NK cells prepared in Example 4 were taken as effector cells.
  • the target cells are K562 cells that do not express HLA-E (human myeloid leukemia cells, Chinese Academy of Sciences Cell Bank), K562 cells that overexpress HLA-E (called K562-HLA-E cells), and FaDu cells that endogenously express HLA-E (human pharyngeal squamous cells, ATCC).
  • HLA-E human myeloid leukemia cells, Chinese Academy of Sciences Cell Bank
  • K562-HLA-E cells K562 cells that overexpress HLA-E
  • FaDu cells human pharyngeal squamous cells, ATCC.
  • the expression levels of HLA-E are shown in Figure 17.
  • the extracellular region of human HLA-E (sequence shown in SEQ ID NO: 78) was transferred into K562 cells by lentivirus-mediated method to obtain K562-HLA-E cells.
  • NK cells 1 ⁇ 10 5 NK cells were co-incubated with the three target cells according to the effect-to-target ratio of 1:1.
  • CD107a-APC antibody purchased from BD Biosciences, 5 ⁇ l/test
  • NKG2A antibody IgG4 form, 10 ⁇ g/ml, blank group without NKG2A antibody
  • protein transport inhibitor Edeldin A/Monensin Mix
  • CD56 antibody with PE-Cy7 fluorescence CD56-PE-Cy7, purchased from eBioscience, 5 ⁇ l/test, used to detect NK cells
  • anti-Fc antibody with FITC fluorescence anti-Fc-FITC antibody, purchased from Jackson Immunoresearch, 1:200, used to detect NKG2A-positive NK cells
  • the blank group was added with CD56-PE-Cy7 and ant After co-incubation with i-NKG2A-PE antibody (purchased from Miltenyi Biotec, 2 ⁇ l/test), FACs detection was performed to analyze the expression of CD107a in NKG2A + NK cells in each group. The results are shown in Figure 18. After co-incubation of NKG2A + NK cells and K562 cells, the expression level of CD107a was higher, indicating that the tumor cell K562 did not bind to NKG2A on NK cells to inhibit the activity of NK cells. Therefore, the addition of NKG2A antibody had no significant effect on the expression level of CD107a.
  • the expression level of CD107a was low, indicating that the combination of HLA-E on tumor cells and NKG2A on NK cells can significantly inhibit the activity of NK cells; adding NKG2A antibody competitively binds to NKG2A on NK cells, blocking the combination of NKG2A on NK cells and HLA-E on tumor cells, thereby reducing HLA- E high-expression tumor cells can inhibit the activity of NK cells, so compared with the group without antibody addition, the expression level of CD107a in each group added with NKG2A antibody was significantly increased. Due to the low endogenous expression of HLA-E in FaDu cells, the increase in CD107a expression level was lower than that in the HLA-E high expression group K562-HLA-E.
  • NKG2A antibody enhances the killing effect of primary NK cells on target cells expressing HLA-E
  • NKG2A + NK cells were co-incubated with target cells (K562, K562-HLA-E and FaDu cells) respectively, with an effect-to-target ratio of 3:1.
  • target cells K562, K562-HLA-E and FaDu cells
  • 10 ⁇ g/ml NKG2A antibody IgG4 format, no antibody was added to the blank group was added. After co-incubating for 4 hours, the supernatant of the medium was collected, and the killing effect was calculated by LDH detection method.
  • Cytotoxicity% [LDH release amount of experimental group (Avg.) - spontaneous LDH release amount of effector cells (Avg.) - target Spontaneous LDH release of cells (Avg.)]/[maximum LDH release of target cells (Avg.)-spontaneous LDH release of target cells (Avg.)-volume calibration (Avg.)] ⁇ 100%
  • NKG2A + NK cells have a killing rate of about 55% on K562 cells, and the killing rates on K562-HLA-E cells and FaDu cells highly expressing HLA-E are lower than 20%. It shows that the HLA-E expressed on the target cell combines with the NKG2A expressed on the NK cell to inhibit the activity of the NK cell, thereby reducing the killing of the target cell by the NK cell.
  • the addition of NKG2A antibody can block the combination of HLA-E of target cells and NKG2A of NK cells, reduce the inhibition of NK cell activity by target cells, thereby increase the activity of NK cells, and increase the ability of NK cells to kill tumor cells with high expression of HLA-E.
  • Example 8 The killing effect of anti-NKG2A specific CAR-T cells on NK cells
  • PRRLSIN-cPPT.EF-1 ⁇ purchased from Addgene
  • PRRLSIN-cPPT.EF-1 ⁇ purchased from Addgene
  • lentiviral plasmids expressing second-generation chimeric antigen receptors of antibodies A4 and A5, namely PRRLSIN-A4-BBZ and PRRLSIN-A5-BBZ were constructed.
  • the A4-BBZ (SEQ ID NO: 115) sequence is composed of CD8 ⁇ signal peptide, A4scFv, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signaling domain and CD3 ⁇ sequentially connected.
  • the A5-BBZ (SEQ ID NO: 116) sequence is composed of CD8 ⁇ signal peptide, A5scFv, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signaling domain and CD3 ⁇ sequentially connected.
  • CD8 ⁇ signal peptide SEQ ID NO: 93
  • A4scFv SEQ ID NO: 64
  • A5scFv SEQ ID NO: 66
  • CD8 hinge region SEQ ID NO: 95
  • CD8 transmembrane region SEQ ID NO: 97
  • CD137 intracellular signaling domain SEQ ID NO: 101
  • CD3 ⁇ SEQ ID NO: 1 05
  • the calcium phosphate method was used to package the lentivirus, and the virus supernatant was purified with PEG8000/NaCl.
  • the purified virus was infected with CD3/CD28 magnetic beads at an MOI of 10 and activated for 48 hours to obtain CAR-T cells expressing A4-BBZ and A5-BBZ. T cells that were not transfected with the virus were regarded as UTD.
  • the CAR positive rate was detected by FACS method.
  • the detection antigen was Bio-NKG2A-CD94, and the secondary antibody was BV421-labeled SA antibody (BD Horizon TM ), diluted at 1:200.
  • the results showed that the CAR positive rate of A4-BBZ CAR T was 62.8%, and that of A5-BBZ CAR T was 59%.
  • NK cells were purified from peripheral blood PBMCs of two donors (#1 and #2) by NK Cell Isolation Kit (Miltenyi Biotec), and treated with NK cells containing 500IU/ml IL-2 and 150IU/ml IL-15 Medium (Miltenyi Biotec) was cultivated until the 14th day and collected.
  • the above-mentioned NK cells were respectively incubated with APC-labeled NKG2A antibody (Invitrogen) (diluted at 1:200) for 5 min at 4°C, and the expression level of NKG2A in NK cells was detected by FACS method.
  • the results showed that the positive rate of NKG2A in NK cells of #1 donor was 80.4%, and the positive rate of NKG2A in NK cells of #2 donor was 61.5%.
  • Target cells 5 ⁇ 10 4 above-mentioned NKG2A positive NK cells were seeded into 96-well plates as target cells.
  • Effector cells Inoculate UTD cells, A4-BBZ CAR-T cells, and A5-BBZ CAR-T cells into corresponding 96-well plates according to the effect-to-target ratio of 1:1 and 2:1, respectively.
  • the in vitro cell killing experiment was carried out by flow cytometry, and flow staining was performed at 0hr, 4hr, and 24hr to detect the proportion of NK cells in the co-culture system.
  • the results are shown in Figure 20.
  • the NK cells in the UTD cell group There was no significant change in the proportion, but the proportion of NK cells in the A4-BBZ CAR-T and A5-BBZ CAR-T groups decreased significantly. This indicates that both A4-BBZ CAR-T cells and A5-BBZ CAR-T cells can effectively kill NKG2A-expressing NK cells.
  • Example 9 Anti-NKG2A UCAR-T cells can effectively resist the killing of NK cells
  • B2M-deleted T cells can cause NK cell rejection, and the resistance of UCAR-T cells to NK cells was verified by constructing TCR/B2M-deleted NKG2A-UCAR-T cells.
  • NKG2A-knockout NKG2A-UCAR-T cells (UCAR-TKO) were prepared at the same time.
  • the gRNA targeting the TCR/B2M/NKG2A gene was synthesized in vitro, and the sequences are shown in SEQ ID NO: 117, 118, and 119, respectively.
  • the endogenous TCR/B2M or TCR/B2M/NKG2A of T cells was knocked out by conventional CRISPR/Cas9 technology.
  • CRISPR/Cas 9 enzyme Kaijia Biology
  • gRNA were mixed at a molar ratio of 1:4 to form an RNP complex (the final concentration of the Cas 9 enzyme was 1uM). After incubation at room temperature for 10 minutes, the RNP complex was introduced into T cells using a MaxCyte electroporator.
  • A4-CAR-T and A5-CAR-T cells in Example 8 were respectively subjected to TCR/B2M double knockout to obtain A4-UCAR-T and A5-UCAR-T cells; A4-CAR-T and A5-CAR-T cells were respectively subjected to TCR/B2M/NKG2A triple knockout to obtain A4-UCAR-T-TKO and A5-UCAR-T-TKO cells.
  • UTD cells (named UTD UCAR-T, UTD UCAR-T-TKO) that knocked out TCR/B2M, TCR/B2M/NKG2A genes in the same way but not transfected with CAR were used as controls, adjusted the cell concentration to 5 ⁇ 10 5 /mL, inoculated into 96-well plates, and inoculated the cells according to the ratio of 1:1 or 1:2 of primary amplified #2 donor NK cells to T cells, and co-incubated in the incubator for 0 hr and 24 hr, respectively. , 48hr. HLA-ABC positive NK cells were labeled by flow cytometry, and the proportion of UCAR-T cells at different time points of co-incubation was detected.
  • BCMA-targeting CAR-T cells Using conventional molecular biology methods in the field, construct a chimeric antigen receptor targeting BCMA, package lentivirus and transfect T cells, and prepare BCMA-targeting CAR-T cells.
  • the amino acid sequence of BCMA-scFv is shown in SEQ ID NO:120
  • the amino acid sequence of BCMA-CAR is shown in SEQ ID NO:121.
  • the B2M/TCR/NKG2A gene of BCMA CAR-T cells was knocked out by the method in Example 9 to obtain BCMA UCAR-T cells (named BCMA UCAR-T-TKO).
  • the BCMA-expressing multiple myeloma cell line RPMI-8226 (Cell Bank, Chinese Academy of Sciences) was cultured in vitro, and 5 ⁇ 10 6 cells per mouse were subcutaneously inoculated into NPG immunodeficient mice (denoted as D0). The average tumor volume was about 200 mm 3 10 days after inoculation, and the mice were divided into 4 groups. On D10, D14, D17, D21, and D24, 1 ⁇ 10 6 NK cells were injected into the tail vein of groups 2, 3, and 4 respectively, for a total of 5 injections. On D11, T cells were injected into the tail vein according to groups. The details of each group are as follows:
  • T cell injection body weight was measured twice a week (including group administration and the day of euthanasia), and the long diameter and short diameter of the tumor were measured and recorded with a caliper, and the tumor volume was calculated.
  • NK cells UCAR-T cells against NKG2A exerted synergistic anti-tumor effects with BCMA UCAR-T cells: on D32, the tumors in the mice in groups 3 and 4 were almost completely eliminated.
  • BCMA-NKG2A CAR-T cells targeting both NKG2A and BCMA were constructed, and their anti-tumor activity and anti-NK cell killing effect were observed.
  • BCMA-NKG2A CAR-T cells expressing tandem CAR (SEQ ID NO: 122) were constructed using the vector PRRLsin.
  • the B2M/TCR/NKG2A gene of BCMA-NKG2A CAR-T cells was knocked out to obtain BCMA-NKG2A UCAR-T-TKO cells.
  • the results of in vitro and in vivo experiments showed that tandem UCAR-T cells targeting NKG2A and tumor antigens could effectively resist the killing of NK cells and inhibit tumor growth.

Abstract

The present invention relates to an antibody targeting NKG2A and an application thereof. Disclosed in the present invention is a novel fully human antibody capable of specifically recognizing NKG2A. The antibody of the present invention can effectively block the binding of HLA-E of tumor cells to NKG2A/CD94 of NK cells, reduces the inhibiting effect of the tumor cells expressing HLA-E on the NK cells by means of NKG2A/CD94 pathway, and enhances the killing effect of the NK cells on the tumor cells.

Description

NKG2A抗体及其应用NKG2A antibody and its application
相关申请related application
本专利申请要求于2022年1月24日递交的申请号为202210080479.9的中国专利申请的优先权。This patent application claims the priority of the Chinese patent application with application number 202210080479.9 submitted on January 24, 2022.
同时提交的序列表文件Sequence listing files submitted at the same time
下列XML文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(名称：FF00718PCT-sequence listing.xml，日期：20230119，大小：148KB)。The entire content of the following XML file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (Name: FF00718PCT-sequence listing.xml, Date: 20230119, Size: 148KB).
技术领域technical field
本发明涉及肿瘤免疫治疗或诊断领域，更具体地，涉及识别NKG2A的抗体及其应用。The present invention relates to the field of tumor immunotherapy or diagnosis, more specifically, to an antibody recognizing NKG2A and its application.
背景技术Background technique
自然杀伤细胞(natural killer cell，NK细胞)是一类无需预先致敏就能非特异性杀伤肿瘤细胞和病毒感染细胞的淋巴细胞，是机体免疫细胞的重要组成之一。NK细胞表面有众多受体，分为激活性和抑制性两类，NKG2A便是其中一类抑制性受体。Natural killer cells (NK cells) are a type of lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization, and are one of the important components of the body's immune cells. There are many receptors on the surface of NK cells, which are divided into two types: activating and inhibitory, and NKG2A is one of the inhibitory receptors.
NKG2A蛋白又叫C型凝集素，不仅表达在NK细胞中，在NKT细胞和CD8+αβT细胞中也有表达，NKG2A可与细胞表面的CD94形成二聚体(Jiacheng Bi and Zhigang Tian.NK Cell Dysfunction and Checkpoint Immunotherapy.Front.Immunol,2019.)。非经典MHC I类分子HLA-E是NKG2A-CD94的主要配体，在正常情况下HLA-E的表达量很低，但是在大部分的肿瘤细胞表面，HLA-E的表达量上升，NKG2A与HLA-E的相互作用抑制了NK细胞的激活，使得肿瘤细胞避免被NK细胞杀伤(Linda Borst,et al.The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment.American Association for Cancer,2020.)。因此开发靶向NKG2A的抗体可阻断NKG2A与HLA-E的结合，进而激活NK细胞和T细胞。NKG2A protein, also called C-type lectin, is expressed not only in NK cells, but also in NKT cells and CD8+αβT cells. NKG2A can form dimers with CD94 on the cell surface (Jiacheng Bi and Zhigang Tian. NK Cell Dysfunction and Checkpoint Immunotherapy. Front. Immunol, 2019.). The non-classical MHC class I molecule HLA-E is the main ligand of NKG2A-CD94. Under normal circumstances, the expression level of HLA-E is very low, but on the surface of most tumor cells, the expression level of HLA-E increases. The interaction between NKG2A and HLA-E inhibits the activation of NK cells, making tumor cells avoid being killed by NK cells (Linda Borst, et al. The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment. American Association for Cancer, 2020.). Therefore, the development of antibodies targeting NKG2A can block the binding of NKG2A to HLA-E, thereby activating NK cells and T cells.
虽然NKG2A抗体极具潜力，但是其开发极具挑战。NKG2受体家族具有多种受体，包括NKG2A，NKG2C，NKG2D和NKG2E等。这些受体对于免疫细胞有些具有抑制作用，有些具有激活作用。这些受体的氨基酸序列具有高度的同源性，其中人的NKG2A和NKG2C同源性为90％，和NKG2E的同源为71％。虽然NKG2C和NKG2E与NKG2A在胞外段序列非常类似，但是在功能上完全相反，对抗体的特异性要求很高。Although NKG2A antibodies have great potential, their development is extremely challenging. The NKG2 receptor family has a variety of receptors, including NKG2A, NKG2C, NKG2D, and NKG2E, among others. Some of these receptors have an inhibitory effect on immune cells, and some have an activating effect. The amino acid sequences of these receptors have high homology, among which the homology of human NKG2A and NKG2C is 90%, and that of NKG2E is 71%. Although NKG2C and NKG2E are very similar to NKG2A in the extracellular sequence, they are completely opposite in function and require high antibody specificity.
目前已有相关NKG2A靶点单克隆抗体药物报道，主要包括Innate/阿斯利康的Monalizumab(US20170298131A1)和怀越生物的Mpb416(CN111153995A)，初步临床结果显示NKG2A抗体联合用药具有一定的抗肿瘤效果，但也存在单药效果较差和具有一定副作用的问题。 At present, relevant NKG2A target monoclonal antibody drugs have been reported, mainly including Innate/AstraZeneca’s Monalizumab (US20170298131A1) and Huaiyue Biotech’s Mpb416 (CN111153995A). Preliminary clinical results show that the combination of NKG2A antibodies has a certain anti-tumor effect, but there are also problems of poor single-drug effect and certain side effects.
发明内容Contents of the invention
本发明的目的在于提供识别NKG2A的全人源抗体及其应用。The purpose of the present invention is to provide a fully human antibody recognizing NKG2A and its application.
在第一方面，本发明提供了一种识别NKG2A的全人源抗体，其特征在于，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYX1X2T(SEQ ID NO:129)所示的LCDR3，其中X1是I或V，X2是R或S。In a first aspect, the present invention provides a fully human antibody that recognizes NKG2A, wherein the antibody comprises a light chain variable region, and the light chain variable region comprises LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYX 1 X 2 T (SEQ ID NO:129), wherein X 1 is I or V, X2 is R or S.
在具体实施方式中，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYIRT(SEQ ID NO:6)所示的LCDR3。In a specific embodiment, the antibody comprises a light chain variable region comprising LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYIRT (SEQ ID NO:6).
在具体实施方式中，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYVST(SEQ ID NO:10)所示的LCDR3。In a specific embodiment, the antibody comprises a light chain variable region comprising LCDR1 shown in RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 shown in DASSLES (SEQ ID NO:5); and/or LCDR3 shown in QQYDSYVST (SEQ ID NO:10).
本发明还提供了一种识别NKG2A的全人源抗体，其特征在于，所述抗体包括重链可变区，所述重链可变区选自：The present invention also provides a fully human antibody that recognizes NKG2A, characterized in that the antibody includes a heavy chain variable region, and the heavy chain variable region is selected from:
(1)包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTAX1YAQKFQG(SEQ ID NO:130)所示的HCDR2，其中X1是N或H；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3；或 or _
(2)包含X1X2X3X4S(SEQ ID NO:131)所示的HCDR1，其中X1是S、R或N，X2是Y、F或V，X3是A、Y或H，X4是M或V；和/或AIX1X2X3X4GSTYYADSVKG(SEQ ID NO:132)所示的HCDR2，其中X1是S、T或N，X2是G或A，X3是S、W、G或P，X4是G或V；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。(2) HCDR1 comprising X 1 X 2 X 3 X 4 S (SEQ ID NO: 131), wherein X 1 is S, R or N, X 2 is Y, F or V, X 3 is A, Y or H, X 4 is M or V; and/or HCDR2 shown in AIX 1 X 2 X 3 X 4 GSTYYADSVKG (SEQ ID NO: 132), wherein X 1 is S, T Or N, X2 is G or A, X3 is S, W, G or P, X4 is G or V; and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTANYAQKFQG(SEQ ID NO:2)所示的HCDR2；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3。In a specific embodiment, the antibody comprises a heavy chain variable region comprising HCDR1 shown in SYAIS (SEQ ID NO:1); and/or HCDR2 shown in GIIPIFGTANYAQKFQG (SEQ ID NO:2); and/or HCDR3 shown in GFDGMDY (SEQ ID NO:3).
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTAHYAQKFQG(SEQ ID NO:11)所示的HCDR2；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3。In a specific embodiment, the antibody comprises a heavy chain variable region comprising HCDR1 shown in SYAIS (SEQ ID NO:1); and/or HCDR2 shown in GIIPIFGTAHYAQKFQG (SEQ ID NO:11); and/or HCDR3 shown in GFDGMDY (SEQ ID NO:3).
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含SYAMS(SEQ ID NO:7)所示的HCDR1；和/或AISGSGGSTYYADSVKG(SEQ ID NO:8)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。In a specific embodiment, the antibody comprises a heavy chain variable region comprising HCDR1 shown in SYAMS (SEQ ID NO:7); and/or HCDR2 shown in AISGSGGSTYYADSVKG (SEQ ID NO:8); and/or HCDR3 shown in GYDGFDY (SEQ ID NO:9).
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含RFYMS(SEQ ID NO:12)所示的HCDR1；和/或AITGWGGSTYYADSVKG(SEQ ID NO:13)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。In a specific embodiment, the antibody comprises a heavy chain variable region comprising HCDR1 shown in RFYMS (SEQ ID NO: 12); and/or HCDR2 shown in AITGWGGSTYYADSVKG (SEQ ID NO: 13); and/or HCDR3 shown in GYDGFDY (SEQ ID NO: 9).
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含RVHMS(SEQ ID NO:14)所示的HCDR1；和/或AISAGGGSTYYADSVKG(SEQ ID NO:15)所示的HCDR2；和/或 GYDGFDY(SEQ ID NO:9)所示的HCDR3。In specific embodiments, the antibody comprises a heavy chain variable region comprising HCDR1 shown in RVHMS (SEQ ID NO: 14); and/or HCDR2 shown in AISAGGGSTYYADSVKG (SEQ ID NO: 15); and/or HCDR3 represented by GYDGFDY (SEQ ID NO:9).
在具体实施方式中，所述抗体包括重链可变区，所述重链可变区包含NFHVS(SEQ ID NO:16)所示的HCDR1；和/或AINGPVGSTYYADSVKG(SEQ ID NO:17)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。In a specific embodiment, the antibody comprises a heavy chain variable region comprising HCDR1 shown in NFHVS (SEQ ID NO: 16); and/or HCDR2 shown in AINGPVGSTYYADSVKG (SEQ ID NO: 17); and/or HCDR3 shown in GYDGFDY (SEQ ID NO: 9).
在具体实施方式中，所述抗体选自以下的任一种：In a specific embodiment, the antibody is selected from any of the following:
(1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1、7、12、14或16所示的HCDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示的HCDR2，和/或包含SEQ ID NO:3或9任一所示的HCDR3；(1) an antibody comprising a heavy chain variable region, the heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising HCDR2 shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and/or comprising HCDR3 shown in any of SEQ ID NO: 3 or 9;
(2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:4所示的LCDR1，和/或包含SEQ ID NO:5所示的LCDR2，和/或包含SEQ ID NO:6或10任一所示的LCDR3；(2) an antibody comprising a light chain variable region comprising LCDR1 shown in SEQ ID NO:4, and/or comprising LCDR2 shown in SEQ ID NO:5, and/or comprising LCDR3 shown in any of SEQ ID NO:6 or 10;
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；(3) an antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。(4) An antibody, a variant of the antibody described in any one of (1) to (3), having the same or similar activity as the antibody described in any one of (1) to (3).
在具体实施方式中，所述抗体选自以下的任一种：In a specific embodiment, the antibody is selected from any of the following:
(1)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或or
(2)抗体，其包含SEQ ID NO:7所示的HCDR1，SEQ ID NO:8所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(2) an antibody comprising HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10;
(3)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:11所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或(3) an antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6;
(4)抗体，其包含SEQ ID NO:12所示的HCDR1，SEQ ID NO:13所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(4) an antibody comprising HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:10; or
(5)抗体，其包含SEQ ID NO:14所示的HCDR1，SEQ ID NO:15所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(5) an antibody comprising HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:10; or
(6)抗体，其包含SEQ ID NO:16所示的HCDR1，SEQ ID NO:17所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；(6) an antibody comprising HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10;
(7)抗体，(1)～(6)中任一项所述的抗体的变体，且具备与(1)～(6)中任一项所述的抗体相同或相似的活性。 (7) An antibody, a variant of the antibody described in any one of (1) to (6), having the same or similar activity as the antibody described in any one of (1) to (6).
在具体实施方式中，所述抗体选自以下的任一种：In a specific embodiment, the antibody is selected from any of the following:
(1)抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:18、22、26、28、30或32所示的氨基酸序列、或上述序列的变体；(1) an antibody comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or a variant of the above sequence;
(2)抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:20或24所示的氨基酸序列、或上述序列的变体；(2) an antibody comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence;
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。(3) An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody.
在具体实施方式中，所述抗体选自以下的任一种：In a specific embodiment, the antibody is selected from any of the following:
(1)抗体，所述抗体的重链可变区具有SEQ ID NO:18所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列；(1) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20;
(2)抗体，所述抗体的重链可变区具有SEQ ID NO:22所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列；(2) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:22, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24;
(3)抗体，所述抗体的重链可变区具有SEQ ID NO:26所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列；(3) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:20;
(4)抗体，所述抗体的重链可变区具有SEQ ID NO:28所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列；(4) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24;
(5)抗体，所述抗体的重链可变区具有SEQ ID NO:30所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列；(5) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24;
(6)抗体，所述抗体的重链可变区具有SEQ ID NO:32所示的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列。(6) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:24.
(7)抗体，(1)～(6)中任一项所述的抗体的变体，且具备与(1)～(6)中任一项所述的抗体相同或相似的活性。(7) An antibody, a variant of the antibody described in any one of (1) to (6), having the same or similar activity as the antibody described in any one of (1) to (6).
在具体实施方式中，第一方面所述的抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或IgG4抗体。In a specific embodiment, the antibody described in the first aspect is a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or IgG4 antibody.
在具体实施方式中，第一方面所述的抗体不显著结合NKG2C、NKG2E或其组合。In a specific embodiment, the antibody of the first aspect does not significantly bind NKG2C, NKG2E or a combination thereof.
在具体实施方式中，第一方面所述的抗体结合NKG2A/CD94，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。In a specific embodiment, the antibody of the first aspect binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94 or a combination thereof.
在具体实施方式中，第一方面所述的抗体结合表达NKG2A/CD94的细胞，不显著结合表达NKG2C/CD94、NKG2E/CD94或其组合的细胞。In a specific embodiment, the antibody of the first aspect binds to cells expressing NKG2A/CD94, but does not significantly bind to cells expressing NKG2C/CD94, NKG2E/CD94 or a combination thereof.
在具体实施方式中，第一方面所述的抗体在降低CD94/NKG2A介导的抑制表达CD94/NKG2A的细胞毒性淋巴细胞的细胞毒性活性中更有效。In a specific embodiment, the antibody described in the first aspect is more effective in reducing CD94/NKG2A-mediated cytotoxicity of CD94/NKG2A-expressing cytotoxic lymphocytes.
在具体实施方式中，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞、NKT细胞、α/βT细胞或γ/δT细胞。In a specific embodiment, the CD94/NKG2A-expressing cytotoxic lymphocytes are NK cells, NKT cells, α/βT cells or γ/δT cells.
在具体实施方式中，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞。In a specific embodiment, said CD94/NKG2A expressing cytotoxic lymphocytes are NK cells.
在第二方面，本发明提供一种免疫缀合物，所述的免疫辍合物包括第一、方面所述的抗 体，以及与之连接的功能性分子。In a second aspect, the present invention provides an immunoconjugate, which includes the anti- body, and functional molecules linked to it.
在第三方面，本发明提供一种嵌合受体，所述嵌合受体的胞外域包含第一方面所述的抗体，所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。In a third aspect, the present invention provides a chimeric receptor, the ectodomain of the chimeric receptor comprises the antibody described in the first aspect, and the chimeric receptor includes: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC) or a combination thereof.
在具体实施方式中，所述嵌合受体是嵌合抗原受体(CAR)。In a specific embodiment, said chimeric receptor is a chimeric antigen receptor (CAR).
在具体实施方式中，所述CAR包含顺序连接的：第一方面所述的抗体、跨膜区和胞内信号区。In a specific embodiment, the CAR comprises sequentially connected: the antibody described in the first aspect, a transmembrane region and an intracellular signal region.
在具体实施方式中，所述的胞内信号区选自：CD3δ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或所述的跨膜区包含CD8或CD28的跨膜区。In a specific embodiment, the intracellular signal region is selected from: the intracellular signal region sequence of CD3δ, FcεRIγ, CD27, CD28, CD137, CD134, MyD88, CD40 or a combination thereof; and/or the transmembrane region comprises the transmembrane region of CD8 or CD28.
在具体实施方式中，所述的CAR包括：第一方面所述的抗体、CD8/CD28的跨膜区和CD3δ；或第一方面所述的抗体、CD8/CD28的跨膜区、CD137的胞内信号区和CD3δ；或第一方面所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区和CD3δ；或第一方面所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3δ。In a specific embodiment, the CAR includes: the antibody of the first aspect, the transmembrane region of CD8/CD28, and CD3δ; or the antibody of the first aspect, the transmembrane region of CD8/CD28, the intracellular signal region of CD137, and CD3δ; or the antibody of the first aspect, the transmembrane region of CD8/CD28, the intracellular signal region of CD28, and CD3δ; or the antibody of the first aspect, the transmembrane region of CD8/CD28, and the intracellular signal region of CD28 , CD137 and CD3δ.
在具体实施方式中，所述的嵌合受体的氨基酸序列如SEQ ID NO：115或116所示。In a specific embodiment, the amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 115 or 116.
在第四方面，本发明提供编码第一方面所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体的核酸。In the fourth aspect, the present invention provides nucleic acid encoding the antibody of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect.
在第五方面，本发明提供一种表达载体，其包含第四方面所述的核酸。In the fifth aspect, the present invention provides an expression vector comprising the nucleic acid described in the fourth aspect.
在第六方面，本发明提供一种病毒，其包含第五方面所述的表达载体或第四方面所述的核酸。In the sixth aspect, the present invention provides a virus comprising the expression vector of the fifth aspect or the nucleic acid of the fourth aspect.
在第四方面、第五方面和第六方面涉及的核酸、表达载体和病毒均为本发明的生物材料。本发明的生物材料，其为如下中的任意一种：The nucleic acids, expression vectors and viruses involved in the fourth, fifth and sixth aspects are all biological materials of the present invention. The biological material of the present invention is any one of the following:
1)编码第一方面的抗体、第二方面的免疫缀合物、第三方面所述的嵌合受体的核酸；1) Nucleic acid encoding the antibody of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect;
2)包含1)所述的表达载体；或者2) comprising the expression vector described in 1); or
3)包含1)或2)所述的病毒。3) comprising the virus described in 1) or 2).
在第七方面，本发明提供一种宿主细胞，所述宿主细胞表达第三方面所述的嵌合受体。In a seventh aspect, the present invention provides a host cell expressing the chimeric receptor of the third aspect.
在具体实施方式中，所述宿主细胞结合表达NKG2A/CD94的细胞，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。In specific embodiments, the host cell binds to cells expressing NKG2A/CD94 and does not significantly bind NKG2C/CD94, NKG2E/CD94, or a combination thereof.
在具体实施方式中，所述宿主细胞能抵抗NK细胞攻击或杀伤NK细胞。In a specific embodiment, the host cell can resist NK cell attack or kill NK cell.
在具体实施方式中，所述宿主细胞还表达识别肿瘤抗原和/或病原体抗原的嵌合受体。In a specific embodiment, the host cell also expresses a chimeric receptor that recognizes tumor antigens and/or pathogen antigens.
在具体实施方式中，所述宿主细胞与靶向肿瘤和/或病原体的第二宿主细胞联合应用。In a specific embodiment, said host cell is used in combination with a second host cell targeting a tumor and/or a pathogen.
在具体实施方式中，所述宿主细胞和/或第二宿主细胞不表达B2M、TCR/B2M、TCR/B2M/CIITA、TCR/B2M/NKG2A、和/或TCR/B2M/CIIA/NKG2A。In specific embodiments, the host cell and/or the second host cell does not express B2M, TCR/B2M, TCR/B2M/CIITA, TCR/B2M/NKG2A, and/or TCR/B2M/CIIA/NKG2A.
在具体实施方式中，所述宿主细胞和/或第二宿主细胞是T细胞、自然杀伤细胞、细胞 毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。In a specific embodiment, the host cell and/or the second host cell is a T cell, a natural killer cell, a cell Toxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, stem cell-derived immune effector cells, or combinations thereof.
在具体实施方式中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞。In a specific embodiment, the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells.
在具体实施方式中，所述T细胞为自体/同种异体T细胞。In a specific embodiment, said T cells are autologous/allogeneic T cells.
在具体实施方式中，所述T细胞为原代T细胞。In a specific embodiment, said T cells are primary T cells.
在具体实施方式中，所述T细胞来源于人的T细胞。In a specific embodiment, the T cells are derived from human T cells.
在具体实施方式中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。In a specific embodiment, the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), γδT cells or combinations thereof.
在第八方面，本发明提供一种联合用药，第一方面所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体、第七方面所述的宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；和/或与改善其相关的一种或多种副作用的药剂联合施用；和/或与表达靶向NKG2A之外的嵌合抗原受体的宿主细胞联合施用。In the eighth aspect, the present invention provides a drug combination, the antibody described in the first aspect, the immunoconjugate described in the second aspect, the chimeric receptor described in the third aspect, and the host cell described in the seventh aspect are administered in combination with agents that enhance their functions, preferably, in combination with chemotherapy drugs; and/or in combination with agents that improve one or more side effects associated therewith; and/or in combination with host cells expressing chimeric antigen receptors targeting NKG2A.
在第九方面，本发明提供一种制备第一方面所述的抗体、第二方面所述的免疫缀合物、第三方面所述的嵌合受体的方法，所述方法包含在适于表达所述抗体、免疫缀合物、嵌合受体的条件下培养第七方面所述的宿主细胞，以及分离出由所述宿主细胞表达的所述抗体、免疫缀合物、组合物、和/或嵌合受体。In the ninth aspect, the present invention provides a method for preparing the antibody of the first aspect, the immunoconjugate of the second aspect, and the chimeric receptor of the third aspect, the method comprising culturing the host cell of the seventh aspect under conditions suitable for expressing the antibody, immunoconjugate, and chimeric receptor, and isolating the antibody, immunoconjugate, composition, and/or chimeric receptor expressed by the host cell.
在第十方面，本发明提供一种药物组合物，其包括：第一方面所述的抗体或编码该抗体的核酸；或第二方面所述的免疫缀合物或编码该缀合物的核酸；或第三方面所述的嵌合受体或编码该嵌合受体的核酸；或第七方面所述的宿主细胞；以及药学上可接受的载体或赋形剂。In the tenth aspect, the present invention provides a pharmaceutical composition, which includes: the antibody described in the first aspect or the nucleic acid encoding the antibody; or the immunoconjugate described in the second aspect or the nucleic acid encoding the conjugate; or the chimeric receptor described in the third aspect or the nucleic acid encoding the chimeric receptor; or the host cell described in the seventh aspect; and a pharmaceutically acceptable carrier or excipient.
在第十一方面，本发明提供一种试剂盒，其包括：In an eleventh aspect, the present invention provides a kit comprising:
容器，以及位于容器中的第十方面所述的药物组合物；或a container, and the pharmaceutical composition of the tenth aspect located in the container; or
容器，以及位于容器中的第一方面所述的抗体或编码该抗体的核酸；或第二方面所述的免疫缀合物或编码该缀合物的核酸；或第三方面所述的嵌合受体或编码该嵌合受体的核酸；或第七方面所述的宿主细胞。A container, and the antibody of the first aspect or the nucleic acid encoding the antibody located in the container; or the immunoconjugate of the second aspect or the nucleic acid encoding the conjugate; or the chimeric receptor of the third aspect or the nucleic acid encoding the chimeric receptor; or the host cell of the seventh aspect.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1显示了利用真核表达质粒V152S构建分别表达NKG2A/CD94、NKG2C/CD94异源二聚体的载体示意图；Figure 1 shows a schematic diagram of constructing vectors expressing NKG2A/CD94 and NKG2C/CD94 heterodimers respectively using the eukaryotic expression plasmid V152S;
图2显示了ELISA检测抗体A1、A2(Fab形式)与NKG2A/CD94、NKG2C/CD94的结合；Figure 2 shows the binding of ELISA detection antibodies A1, A2 (Fab form) to NKG2A/CD94, NKG2C/CD94;
图3显示了ELISA检测抗体A1、A2(IgG4形式)结合NKG2A/CD94的EC50； Figure 3 shows the EC50 of ELISA detection antibody A1, A2 (IgG4 form) binding to NKG2A/CD94;
图4显示了Biacore检测抗体A1、A2(IgG4形式)的亲和力；Fig. 4 shows the affinity of Biacore detection antibody A1, A2 (IgG4 form);
图5显示了带Flag的目的基因CD94-Flag、NKG2A-CD94-Flag、NKG2C-CD94-Flag和NKG2E-CD94-Flag的载体图；Figure 5 shows the vector map of the target gene CD94-Flag, NKG2A-CD94-Flag, NKG2C-CD94-Flag and NKG2E-CD94-Flag with Flag;
图6显示了FACs检测抗体A1、A2(IgG4形式)与CHOK1-NKG2A-CD94、CHOK1-NKG2C-CD94、CHOK1-NKG2E-CD94和CHOK1-CD94细胞的结合；Figure 6 shows the binding of FACs detection antibodies A1, A2 (IgG4 form) to CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94, CHOK1-NKG2E-CD94 and CHOK1-CD94 cells;
图7显示了抗体A1、A2、A3、A4、A5、A6的重链可变区的氨基酸序列对比；Figure 7 shows the amino acid sequence comparison of the heavy chain variable regions of antibodies A1, A2, A3, A4, A5, and A6;
图8显示了FACs检测抗体A1、A2、A3、A4、A5、A6(Fab形式)与CHOK1-NKG2A-CD94、CHOK1-NKG2C-CD94和CHOK1-NKG2E-CD94细胞的结合；Figure 8 shows the binding of FACs detection antibodies A1, A2, A3, A4, A5, A6 (Fab form) to CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94 and CHOK1-NKG2E-CD94 cells;
图9显示了ELISA检测抗体A1、A2、A3、A4、A5、A6(Fab形式)与NKG2A/CD94异源二聚体结合的EC50；Figure 9 shows the EC50 of ELISA detection antibodies A1, A2, A3, A4, A5, A6 (Fab format) combined with NKG2A/CD94 heterodimer;
图10显示了FACs检测抗体A1、A2、A3、A4、A5、A6(Fab形式)与过表达NKG2A/CD94异源二聚体的CHO-K1细胞结合的EC50；Figure 10 shows the EC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (Fab form) combined with CHO-K1 cells overexpressing NKG2A/CD94 heterodimer;
图11显示了ELISA检测抗体A1、A2、A3、A4、A5、A6(IgG4形式)与NKG2A/CD94异源二聚体结合的EC50；Figure 11 shows the EC50 of ELISA detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) combined with NKG2A/CD94 heterodimer;
图12显示了FACs检测抗体A1、A2、A3、A4、A5、A6(IgG4形式)与过表达NKG2A/CD94的CHOK1细胞的EC50；Figure 12 shows the EC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) and CHOK1 cells overexpressing NKG2A/CD94;
图13显示了Biacore检测抗体A3、A4、A5、A6(IgG4形式)的亲和力；Figure 13 shows the affinity of Biacore detection antibodies A3, A4, A5, A6 (IgG4 form);
图14显示了pET22b-HLA-E和pET22b-β2m的载体图；Figure 14 shows the vector maps of pET22b-HLA-E and pET22b-β2m;
图15显示了HLA-E四聚体与表达NKG2A/CD94的NK细胞结合的EC50；Figure 15 shows the EC50 of HLA-E tetramer binding to NK cells expressing NKG2A/CD94;
图16显示了FACs检测抗体A1、A2、A3、A4、A5、A6(IgG4形式)阻断NKG2A与其配体HLA-E结合的IC50；Figure 16 shows the IC50 of FACs detection antibodies A1, A2, A3, A4, A5, A6 (IgG4 form) blocking the combination of NKG2A and its ligand HLA-E;
图17显示了肿瘤细胞K562-HLA-E、K562和FaDu细胞上的HLA-E表达水平；Figure 17 shows the expression levels of HLA-E on tumor cells K562-HLA-E, K562 and FaDu cells;
图18显示了FACs检测NK细胞与K562、K562-HLA-E、FaDu细胞共孵育及加入抗体A1、A2、A3、A4、A5、A6(IgG4形式)后CD107a的表达情况；Figure 18 shows the expression of CD107a detected by FACs after the co-incubation of NK cells with K562, K562-HLA-E, FaDu cells and the addition of antibodies A1, A2, A3, A4, A5, A6 (IgG4 form);
图19显示了FACs检测抗NKG2A抗体对NK细胞在K562、K562-HLA-E、FaDu细胞上发挥杀伤作用的影响；Figure 19 shows the effects of FACs detection of anti-NKG2A antibodies on the killing effect of NK cells on K562, K562-HLA-E, and FaDu cells;
图20显示了A4-BBZ CAR T细胞、A5-BBZ CAR T细胞与NK细胞共孵育后NK细胞比例变化。Figure 20 shows the changes in the proportion of NK cells after co-incubation of A4-BBZ CAR T cells, A5-BBZ CAR T cells and NK cells.
图21显示了A4、A5 UCAR-T与NK细胞共孵育后UCAR-T细胞比例变化。Figure 21 shows the changes in the ratio of UCAR-T cells after co-incubation of A4 and A5 UCAR-T cells with NK cells.
图22显示了A4、A5-UCAR-T与BCMA UCAR-T在NK细胞存在下联合作用于RPMI-8226细胞皮下移植瘤的情况。Figure 22 shows the joint action of A4, A5-UCAR-T and BCMA UCAR-T in the presence of NK cells for subcutaneous xenografts of RPMI-8226 cells.
具体实施方式Detailed ways
本发明人经过深入的研究筛选，获得了特异性识别NKG2A的全人源抗体，包括Fab形式 的抗体、IgG4形式的抗体。本发明的抗体可以被应用于制备靶向抗肿瘤药物以及诊断肿瘤的药物。After in-depth research and screening, the inventors obtained a fully human antibody specifically recognizing NKG2A, including the Fab form Antibodies in IgG4 form. The antibody of the present invention can be applied to the preparation of targeted anti-tumor drugs and drugs for diagnosing tumors.
术语the term
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗、生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, in which case suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting unless otherwise specified.
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrooke et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis(M.J.Gaited., 1984); Mullis et al.U.S.Pat.No.4,683,195; Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984);Transcription And Translation(B.D.Hames&S.J.Higginseds.1984); Culture Of Animal Cells(R.I.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes(IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning(1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols.154 and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); Gene Transfer Vectors For Ma mmalian Cells (J.H.Miller and M.P.Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
公开内容中，请求保护的主题的各个方面均以范围形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。这适用而无关范围的宽度。 In this disclosure, various aspects of claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual values within that range. For example, where a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range, as well as any other stated or intervening value within the range, is encompassed within the claimed subject matter, and that the upper and lower limits of the range are also within the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the stated smaller ranges, and are also within the scope of the claimed subject matter unless the upper and lower limits of the stated range are expressly excluded. Where the stated range includes one or both of the limits, claimed subject matter also includes ranges excluding either or both of those limits. This applies regardless of the width of the range.
术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”或“包含”应假定为在该特定值或组成的可接受误差范围内。The term "about" refers to the usual error range for each value readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments referring to that value or parameter itself. For example, description of "about X" includes description of "X." For example, "about" or "comprising" can mean within 1 or more than 1 standard deviation as actual in the art. Alternatively "about" or "comprising" can mean a range of up to 10% (ie, ±10%). For example, about 5 uM can include any number between 4.5 uM and 5.5 uM. Where specific values or compositions are provided in the applications and claims, unless otherwise indicated, "about" or "comprising" should be assumed to be within an acceptable error range for the specific value or composition.
除非另外指出，本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值。Unless otherwise indicated, any concentration range, percentage range, ratio range or integer range recited herein is to be understood as including any integer and, where appropriate, fractional values (eg, tenths and hundredths of an integer) thereof within the stated range.
为了更易于理解本发明，首先定义一些术语。In order to make the present invention easier to understand, some terms are first defined.
术语“NKG2A”(Natural killer group 2A)是NKG2凝集素受体家族中抑制性的受体，也被称为自然杀伤细胞凝集素样受体C1(killer cell lectin like receptor C1,KLRC1),CD159a、NK细胞受体A，是NK细胞表面优先表达的跨膜蛋白之一。NKG2A主要表达在NK细胞表面和部分T细胞(CD8+T细胞、Th2细胞、γδT细胞以及NKT细胞)。在人的免疫细胞表面,NKG2A与CD94分子(NK细胞表面膜蛋白)以二硫键连接形式形成的异源二聚体NKG2A-CD94,被靶细胞上非经典的组织相容性复合体I(major histocompatibility complex class I,MHC I)类分子HLA-E识别,该分子在正常情况下低表达，但在大部分的肿瘤细胞表面，HLA-E的表达量上升，从而诱导级联的抑制信号,抑制NK的细胞毒活性和细胞因子的分泌。某些病毒感染，肿瘤和免疫性疾病通过该途径逃避免疫检查。NKG2A与HLA-E的相互作用能抑制NK细胞和T细胞的激活。NK细胞经IL15活化后高表达NKG2A。NKG2家族还包括NKG2C、NKG2D和NKG2E。The term "NKG2A" (Natural killer group 2A) is an inhibitory receptor in the NKG2 lectin receptor family, also known as natural killer cell lectin like receptor C1 (KLRC1), CD159a, NK cell receptor A, and is one of the transmembrane proteins preferentially expressed on the surface of NK cells. NKG2A is mainly expressed on the surface of NK cells and some T cells (CD8+T cells, Th2 cells, γδT cells and NKT cells). On the surface of human immune cells, the heterodimer NKG2A-CD94 formed by NKG2A and CD94 molecules (NK cell surface membrane protein) in the form of disulfide bonds is recognized by the non-classical histocompatibility complex I (major histocompatibility complex class I, MHC I) molecule HLA-E on the target cells. The expression of this molecule is low under normal conditions, but on the surface of most tumor cells, the expression of HLA-E increases , thereby inducing a cascade of inhibitory signals, inhibiting the cytotoxic activity of NK and the secretion of cytokines. Certain viral infections, tumors and immune diseases escape immune inspection through this pathway. The interaction between NKG2A and HLA-E can inhibit the activation of NK cells and T cells. NKG2A is highly expressed in NK cells activated by IL15. The NKG2 family also includes NKG2C, NKG2D and NKG2E.
“NKG2A”包括来自任何脊椎动物来源，包括哺乳动物如灵长类(例如，人和猴)和啮齿类(例如，小鼠和大鼠)的任何天然NKG2A。该术语包括“全长”未加工的NKG2A以及来源于细胞中的加工的任何形式的NKG2A。该术语还包括天然存在的NKG2A的变体，例如剪接变体或等位变体。在一实施例中，本文描述的抗NKG2A抗体抑制NKG2A蛋白与HLA-E的结合，因此起到检查点抑制剂的作用。示例性，人NKG2A全长的氨基酸序列如SEQ ID NO:68所示，NKG2A胞外段的氨基酸序列如SEQ ID NO:70所示，人NKG2C全长的氨基酸序列如SEQ ID NO:123所示,NKG2C胞外段的氨基酸序列如SEQ ID NO:72所示，人NKG2E全长的氨基酸序列如SEQ ID NO:125所示,NKG2E胞外段的氨基酸序列如SEQ ID NO:74所示。"NKG2A" includes any native NKG2A from any vertebrate source, including mammals such as primates (eg, humans and monkeys) and rodents (eg, mice and rats). The term includes "full length" unprocessed NKG2A as well as any form of NKG2A derived from processing in the cell. The term also includes naturally occurring variants of NKG2A, such as splice variants or allelic variants. In one embodiment, the anti-NKG2A antibodies described herein inhibit the binding of NKG2A protein to HLA-E, thereby functioning as checkpoint inhibitors. Exemplarily, the amino acid sequence of the full-length human NKG2A is shown in SEQ ID NO:68, the amino acid sequence of the extracellular segment of NKG2A is shown in SEQ ID NO:70, the amino acid sequence of the full-length human NKG2C is shown in SEQ ID NO:123, the amino acid sequence of the extracellular segment of NKG2C is shown in SEQ ID NO:72, and the amino acid sequence of the full-length human NKG2E is shown in SEQ ID NO:125. The amino acid sequence of the extracellular segment of KG2E is shown in SEQ ID NO:74.
术语“人类白细胞抗原”(Human leukocyte antigen，HLA)是人类的主要组织相容性复合体MHC的编码基因，与人类的免疫系统功能密切相关。HLA包括有I类、II类和III类基因部分。HLA的I类和II类基因所表达的抗原位于细胞膜上，为MHC-I(HLA-A、HLA-B、HLA-C位点编码)和MHC-II(HLA-D区编码)，HLA I类几乎分布于身体全部细胞表面，是一个异二聚体，由重链(α链)与β2微球蛋白组成(B2M)，II类主要是定位于巨噬 细胞和B淋巴细胞表面的糖蛋白。The term "Human leukocyte antigen" (Human leukocyte antigen, HLA) is the coding gene of the human major histocompatibility complex MHC, which is closely related to the function of the human immune system. HLA includes class I, class II and class III gene portions. The antigens expressed by HLA class I and class II genes are located on the cell membrane and are MHC-I (encoded by HLA-A, HLA-B, HLA-C sites) and MHC-II (encoded by HLA-D region). HLA class I is distributed on the surface of almost all cells in the body. It is a heterodimer composed of heavy chain (α chain) and β2 microglobulin (B2M). Class II is mainly located in macrophages Glycoproteins on the surface of cells and B lymphocytes.
术语“HLA-E”(OMIM 143010、基因编号NM_005516.6)是非经典MHC分子，其在细胞表面表达，并且通过肽(例如源于其他MHC I类分子的信号序列的片段)的结合而被调节。HLA-E通过特异性结合CD94/NKG2A、CD94/NKG2B和CD94/NKG2C(参见，例如，布劳德(Braud)等人，(1998)自然(Nature)391：795-799，其全部公开内容通过引用并入本文)而结合自然杀伤(NK)细胞、自然杀伤T细胞(NKT)和T细胞(α/β和γ/δ)的亚群。细胞表面表达HLA-E的靶细胞免受CD94/NKG2A阳性的NK、T、或NKT细胞克隆的裂解。如本文所使用的，“HLA-E”是指HLA-E基因或编码的蛋白的任何变体、衍生物或同种型。人HLA-E胞外区氨基酸序列如SEQ ID NO:78所示。HLA-E广泛低水平分布于全身细胞。在几种肿瘤发现高水平的HLA-E，包括妇科肿瘤(高达90％的肿瘤样本)，以及高达50％的乳腺癌、非小细胞肺癌(NSCLC)、肝脏、胰腺、肾脏、黑色素瘤、前列腺、头颈部、胃、直肠和结肠直肠癌。(《The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment》Clin Cancer Res.2020 Nov 1；26(21):5549-5556.)。HLA-E高表达肿瘤通过与免疫细胞(如NK细胞、T细胞)的NKG2A结合来逃逸免疫细胞杀伤，本发明公开的NKG2A抗体能够抑制HLA-E高表达肿瘤免疫逃逸从而杀伤该肿瘤细胞。The term "HLA-E" (OMIM 143010, gene number NM_005516.6) is a non-classical MHC molecule expressed on the cell surface and regulated by the binding of peptides such as fragments of signal sequences derived from other MHC class I molecules. HLA-E through specific binding CD94/NKG2A, CD94/NKG2B, and CD94/NKG2C (see, for example, Braud and others, (1998) Nature 391: 795-799, all of which are combined with increasing this article by citing this article), and natural killing (NK) cells, natural killing killings) T cells (NKT) and sub -groups of T cells (α/β and γ/Δ). Target cells expressing HLA-E on the cell surface are protected from lysis by CD94/NKG2A-positive NK, T, or NKT cell clones. As used herein, "HLA-E" refers to any variant, derivative or isoform of the HLA-E gene or encoded protein. The amino acid sequence of human HLA-E extracellular region is shown in SEQ ID NO:78. HLA-E is widely distributed in low-level cells throughout the body. High levels of HLA-E are found in several tumors, including gynecologic tumors (up to 90% of tumor samples), and up to 50% of breast, non-small cell lung (NSCLC), liver, pancreas, kidney, melanoma, prostate, head and neck, stomach, rectal, and colorectal cancers. ("The NKG2A-HLA-E axis as a novel checkpoint in the tumor microenvironment" Clin Cancer Res.2020 Nov 1; 26(21):5549-5556.). Tumors with high HLA-E expression escape immune cell killing by combining with NKG2A of immune cells (such as NK cells and T cells). The NKG2A antibody disclosed in the present invention can inhibit the immune escape of tumors with high HLA-E expression to kill the tumor cells.
术语“溶酶体相关膜蛋白1(CD107a)”，是囊泡膜蛋白的主要成分，在细胞胞浆中主要构成以囊泡形式存在的细胞毒性颗粒。NK细胞杀伤靶细胞时，释放的细胞毒性颗粒将到达靶细胞膜并与靶细胞膜融合，引起颗粒内容物释放，最终导致靶细胞的死亡。静息状态下，NK细胞膜表面CD107a自发表达率很低，经靶细胞刺激后可以在其表面检测到CD107a表达的增加，因此，NK细胞受刺激后CD107a分子增加的幅度可反映NK细胞的细胞毒性细胞杀伤活性水平。The term "lysosome-associated membrane protein 1 (CD107a)" is the main component of vesicle membrane protein, and mainly constitutes cytotoxic granules in the form of vesicles in the cytoplasm of cells. When NK cells kill target cells, the released cytotoxic granules will reach the target cell membrane and fuse with the target cell membrane, causing the release of the granule contents, eventually leading to the death of the target cell. In the resting state, the spontaneous expression rate of CD107a on the surface of NK cell membrane is very low, and the increase of CD107a expression can be detected on the surface after stimulation of target cells. Therefore, the increase of CD107a molecule after NK cell stimulation can reflect the level of cytotoxic cell killing activity of NK cells.
术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换使用，指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。The terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, cyclic or branched, it may comprise modified amino acids, especially conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes modified amino acid polymers such as amino acid polymers that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenonylation, transfer-RNA-mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with a labeling component, etc. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed from a specified nucleic acid sequence.
术语“抗体”在本文中以最广义使用并且包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要其显示所需的抗原结合活性即可。The term "antibody" is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
“抗体片段”是指不同于完整抗体的分子，其包含完整抗体结合完整抗体所结合的抗原的 部分。抗体片段的实例包括但不限于(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341:544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段；和(ix)与相同或不同抗体遗传融合的scFv。"Antibody fragment" refers to a molecule other than an intact antibody that contains a fragment of the intact antibody in combination with the antigen to which the intact antibody binds. part. Examples of antibody fragments include, but are not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F( ab') 2 fragments, bivalent fragments comprising 2 linked Fab fragments; (vi) antigen binding sites of single chain Fv molecules; (vii) bispecific single chain Fv dimers (PCT/US92/09965); (viii) "dimers" or "trimers", multivalent or multispecific fragments constructed by genetic fusion; and (ix) scFv genetically fused to the same or different antibodies.
抗体的“分类”是指其重链所具有的恒定结构域或恒定区的类型。主要有五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以被进一步划分成亚类(同种异型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同的类型的免疫球蛋白的重链恒定结构域被分别称为α，δ，ε，γ，和μ。The "class" of an antibody refers to the type of constant domain or region that its heavy chain possesses. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (allotypes), eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
术语“可变区或可变结构域”是指参与抗体抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链可变结构域(分别为VH和VL)通常具有相似的结构，其中各结构域包含四个保守的FR和三个CDR(参见，例如，Kindt等，KubyImmunology,6th ed.,W.H.Freeman&Co.,第91页(2007))。单个VH或VL结构域可足以给予抗原结合特异性。此外，结合特定抗原的抗体可以分别使用来自与所述抗原结合的抗体的VH或V L结构域筛选互补VL或VH结构域的文库来分离。参见，例如，Portolano等,J.Immunol.150:880-887(1993)；Clarkson等,Nature352:624-628(1991)。The term "variable region or variable domain" refers to the domains of an antibody heavy or light chain that participate in antibody antigen binding. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies typically have similar structures, with each domain comprising four conserved FRs and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using the VH or VL domains from antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
术语“高变区”或“互补决定区”或“CDR”是指抗体可变结构域中序列高变和/或形成结构确定的环(“高变环”)和/或含有与抗原接触的残基(“抗原触点”)的各区域。通常，抗体包含六个CDR：VH中的三个(HCDR1，HCDR2，HCDR3)和VL中的三个(LCDR1，LCDR2，LCDR3)。The term "hypervariable region" or "complementarity determining region" or "CDR" refers to regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops") and/or contain residues that make contact with an antigen ("antigen contacts"). Typically, antibodies contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3).
术语“Fc区”或“Fc”被用于限定含有恒定区的至少一部分的免疫球蛋白重链的C-端区域。该术语包括天然序列Fc区和变体Fc区。The term "Fc region" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.
“框架(FR)”是指不同于高变区(CDR)残基的可变结构域残基。可变结构域的FR通常由四个FR结构域组成：FR1，FR2，FR3和FR4。因此，在VH(或VL)中CDR和FR序列通常按以下顺序出现：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。"Framework (FR)" refers to variable domain residues that are distinct from hypervariable region (CDR) residues. The FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, in VH (or VL) the CDR and FR sequences typically appear in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.
除非另外指出，在本文中，CDR残基和可变结构域中的其他残基(例如，FR残基)根据以上Kabat等编号。Unless otherwise indicated, herein, CDR residues and other residues in the variable domain (eg, FR residues) are numbered according to Kabat et al. above.
术语“天然抗体”是指天然存在的具有多种结构的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异源四聚糖蛋白，由通过二硫键键合的两个相同的轻链和两个相同的重链组成。从N端到C端，各重链具有可变区(VH)，其也被称为可变重链结构域或重链可变结构域，之后是三个恒定结构域(CH1，CH2和CH3)。类似地，从N端到C端，各轻链具有可变区(VL)，其也被称为可变轻链结构域或轻链可变结构域，之后是轻链恒定(CL)结构域。抗体的轻链基于其恒定结构域的氨基酸序列可以被分配至两种类型之一，被称为κ(κ)和λ(λ)。The term "natural antibody" refers to naturally occurring immunoglobulin molecules of various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a light chain constant (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.
术语“全抗”、“全长抗体”、“完整抗体”可交换使用，是指具有与天然抗体结构基本类似的 结构或具有含有如本文中所限定的Fc区的重链或包括具有抗原结合区域的完整的全长抗体。The terms "full antibody", "full-length antibody" and "intact antibody" are used interchangeably and refer to a protein having a structure substantially similar to a natural antibody. The structure either has a heavy chain comprising an Fc region as defined herein or comprises a complete full length antibody with an antigen binding region.
术语“单域抗体(Single domain antibody,sdAb)”是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。The term "single domain antibody (sdAb)" refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called a nanobody (Nanobody).
术语“单结构域抗体”是指包含抗体的全部或部分的重链可变结构域或全部或部分的轻链可变结构域。在某些实施方案中，单结构域抗体是人单结构域抗体(Domantis,Inc.,Waltham,MA；参加，例如，美国专利号6248516)。The term "single domain antibody" refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, US Patent No. 6248516).
术语“单克隆抗体”、“单抗”是指获自基本上同源的抗体的群体的抗体，即，包括所述群体的个体抗体是相同的和/或结合相同的表位，除可能的变体抗体以外，例如，含天然存在的突变或在单克隆抗体制剂的制备过程中产生，此种变体通常少量存在。对比于多克隆抗体制剂(通常包括针对不同的决定簇(表位)的不同抗体)，单克隆抗体制剂中的各单克隆抗体是针对抗原上的单个决定簇。因此，定于“单克隆”指示抗体的性质为获得自基本上同源的抗体群体，并且不视为要求通过任何特定的方法制备所述抗体。例如，可以通过多种技术制备，包括但不限于杂交瘤方法、重组DNA法、噬菌体展示法，以及利用含有所有或部分的人免疫球蛋白基因座的转基因动物的方法。The terms "monoclonal antibody", "mAb" refer to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during the preparation of monoclonal antibody preparations, such variants usually being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Accordingly, the designation "monoclonal" indicates that the nature of the antibody is that it was obtained from a substantially homogeneous population of antibodies, and is not considered to require that the antibody be prepared by any particular method. For example, they can be produced by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.
术语“嵌合抗体”是指抗体重链和/或轻链的一部分来源于特定来源或物种，而重链和/或轻链的剩余部分来源于不同的来源或物种的抗体。在某些实施方案中，嵌合抗体包含非人可变区(例如，来源于小鼠、大鼠、仓鼠、兔或非人灵长类动物如猴的可变区)和人恒定区。在另外的实施方案中，嵌合抗体是“类型转换”抗体，其中类型或亚类已经由亲本抗体的类型或亚类改变。嵌合抗体包括其抗原结合片段。在某些实施方案中，嵌合抗体是“人源化抗体”。The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain of an antibody is derived from a particular source or species, and the remaining portion of the heavy and/or light chain is derived from a different source or species. In certain embodiments, chimeric antibodies comprise non-human variable regions (eg, variable regions derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and human constant regions. In additional embodiments, chimeric antibodies are "class-switched" antibodies, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, chimeric antibodies are "humanized antibodies."
术语“人源化”用于非人抗体，例如啮齿动物或灵长类动物等，是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。“人源化抗体”是指一种嵌合抗体，其包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体将包含基本上全部的至少一个(一般为两个)可变结构域，其中所有或基本上所有的CDR对应于非人抗体的CDR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。The term "humanized" is used for non-human antibodies, such as rodents or primates, etc., that are hybrid immunoglobulins, immunoglobulin chains or fragments thereof that contain minimal sequence derived from non-human immunoglobulins. A "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (typically two) variable domains in which all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
在一些实施方案中，“人源化抗体”可包括突变，例如通过体外随机或定点诱变或通过体内体细胞突变引入的突变。In some embodiments, a "humanized antibody" may include mutations, for example, introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
术语“全人源抗体”是一种抗体，其具有的氨基酸序列对应于由人或人细胞产生的抗体或来源于利用人抗体库或其他人抗体编码序列的非人来源的抗体的氨基酸序列。全人源体的定义明确排除了包含非人抗原结合残基的人源化抗体。在某些实施方案中，本文中提供的抗体是“全人源抗体”，是由噬菌体展示技术生成。The term "fully human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using human antibody repertoires or other human antibody coding sequences. The definition of fully human specifically excludes humanized antibodies that contain non-human antigen-binding residues. In certain embodiments, the antibodies provided herein are "fully human antibodies" generated by phage display technology.
本发明的抗体可以通过筛选具有所需一种或多种活性的抗体的组合文库来分离。例如，本领域中已知多种方法用于生成噬菌体展示文库并且针对具有所需结合性质的抗体筛选所述文 库。此种方法被综述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，Human Press,Totowa,NJ,2001)中并被进一步描述于例如McCafferty等，Nature 348：552-554；Clackson等，Nature 352：624-628(1991)；Marks等，J.Mol.Biol.222:581-597(1992)；Marks,Meth.Mol.Biol.,248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等，J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA101(34):12467-12472(2004)；Lee等，J.Immunol.Methods 284(1-2):119-132(2004)中。Antibodies of the invention can be isolated by screening combinatorial libraries of antibodies possessing the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening the libraries for antibodies with desired binding properties. library. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J .Mol.Biol.222:581-597 (1992); Marks, Meth.Mol.Biol., 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J.Mol.Biol.338(2):299-310 (2004); Lee et al., J.Mol.Biol.340( 5): 1073-1093 (2004); Fellouse, Proc.
在某些噬菌体展示方法中，VH和VL基因库通过聚合酶链式反应(PCR)分别克隆并且在噬菌体文库中随机重组，然后可以针对结合抗原的噬菌体筛选所述文库，如Winter等，Ann.Rev.Immunol.12:433-455(1994)中所述。噬菌体典型地将抗体片段展示位单链Fv(scFv)片段或Fab片段。来自被免疫的来源的文库向免疫原提供高亲和力抗体而不需要构建杂交瘤。备选地，可以克隆天然库(例如，由人)从而向多种非自身抗原以及自身抗原提供单一来源的抗体而不需要进行任何免疫，如Griffiths等，EMBO J,12:725-734(1993)所述。最后，也可以通过以下方式合成制备天然文库：自干细胞克隆未重排的V-基因区段，并且使用含随机序列的PCR引物以编码高变CDR3区并且在体外实现重排，如Hoogenboom，J.Mol.Biol.227:381-388(1992)所述。In certain phage display methods, VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural Libraries (eg, from humans) thus provide a single source of antibodies to multiple non-self as well as self antigens without requiring any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 region and effecting the rearrangement in vitro as described by Hoogenboom, J. Mol. Biol. 227:381-388 (1992).
在本文中，分离自全人源抗体文库的抗体或抗体片段被认为是全人源抗体或全人源抗体片段。Herein, antibodies or antibody fragments isolated from fully human antibody libraries are considered fully human antibodies or fully human antibody fragments.
在某些实施方案中，本文中提供抗体的氨基酸序列变体。术语“亲本抗体”指本申请所提供的抗体或依据本申请所提供的抗体进行突变、或亲和力成熟等处理后所得的抗体。所述亲本抗体可以是天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。In certain embodiments, provided herein are amino acid sequence variants of the antibodies. The term "parental antibody" refers to the antibody provided in this application or the antibody obtained after undergoing mutation or affinity maturation according to the antibody provided in this application. The parent antibody may be a naturally occurring antibody, or a variant or engineered version of a naturally occurring antibody. A parent antibody may refer to the antibody itself, a composition comprising said parent antibody, or an amino acid sequence encoding it.
术语“亲和力成熟”抗体是指相比于亲本抗体在一个或多个高变区(HVR)中具有一个或多个改变的抗体，此种改变导致抗体对抗原的亲和力提高。The term "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to the parent antibody, such alterations resulting in an increase in the affinity of the antibody for the antigen.
术语“变体”指与本申请所提供的抗体的序列具有基本上相同氨基酸序列或由基本上相同的核苷酸序列编码的一种或多种活性的多肽。所述变体与本申请实施例中所提供的抗体具有相同或相似的活性。The term "variant" refers to a polypeptide having substantially the same amino acid sequence or one or more activities encoded by the substantially same nucleotide sequence as the sequence of the antibody provided herein. The variant has the same or similar activity as the antibody provided in the examples of the present application.
术语“变体抗体”或“抗体变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％％、91％、92％、93％、94％、95％％、96％、97％、98％、99％的氨基酸序列同一性。抗体变体可以指抗体本身，也可以指包含所述抗体变体的组合物。抗体的氨基酸序列变体可以通过向编码所述抗体的核苷酸序列引入合适的修饰或通过肽合成来制备。术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”、“氨基酸置换”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸，“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸，“氨基酸缺失”意指去除亲本多肽序列中特定位置 上的氨基酸。可以进行缺失、插入和取代的任意组合以获得最终的构建体，前提是最终的构建体具有所需的特征，例如：结合抗原。The term "variant antibody" or "antibody variant" includes antibody sequences that differ from a parent antibody sequence by at least one amino acid modification compared to the parent. The variant antibody sequences herein preferably have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity to the parent antibody sequence. Antibody variants may refer to the antibody itself or to a composition comprising the antibody variant. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. The term "amino acid modification" includes amino acid substitution, addition and/or deletion, "amino acid substitution" and "amino acid replacement" mean replacing an amino acid at a specific position in a parental polypeptide sequence with another amino acid, "amino acid insertion" means adding an amino acid at a specific position in a parental polypeptide sequence, and "amino acid deletion" means removing a specific position in a parental polypeptide sequence amino acids on. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct provided that the final construct possesses the desired characteristics, eg binding of antigen.
术语“修饰”一词是指本发明的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。The term "modification" refers to a change in the state or structure of the protein or polypeptide of the present invention. Modifications can be chemical, structural and functional.
术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变、PCR介导的诱变。本领域已经定义了具有相似侧链的氨基酸残基家族，如表1所示。The term "conservative modification" or "conservative sequence modification" means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis. Families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.
表1：具有相似侧链的氨基酸残基家族
Table 1: Families of amino acid residues with similar side chains
因而，可以用其他相同侧链家族的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。Thus, one or more amino acid residues in the CDR regions or in the framework regions of an antibody of the invention can be replaced with other amino acid residues of the same side chain family, and the altered antibody (variant antibody) can be tested for retained function.
非保守置换需要将这些组之一的成员换成另一组的成员。Non-conservative substitutions entail exchanging a member of one of these groups for a member of another group.
一种置换变体包括置换亲本抗体(例如，人源化或人抗体)的一个或多个高变区残基。通常，被选择用于进一步研究的所得的变体相对于亲本抗体将具有某些生物学性质(例如，增加的亲和力、减小的免疫原性)的改变(例如，提高)和/或将基本上保持亲本抗体的某些生物学性质。示例性替换变体是亲和力成熟抗体，其可以例如，使用基于噬菌体展示的亲和力成熟技术(如本文中描述的那些)常规制备。简言之，将一个或多个CDR残基突变并将变体抗体展示在噬菌体上并且筛选特定的生物学活性(例如，结合亲和力)。A substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variant selected for further study will have an altered (e.g., improved) certain biological property (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which can be routinely prepared, eg, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for specific biological activity (eg, binding affinity).
改变(例如，置换)可以在CDR区进行，例如，以提高抗体亲和力。此种改变可以在HVR“热点”中进行，所述“热点”即在体细胞成熟过程期间以高频率进行突变的密码子编码的残基(参见，例如，Chowdhury,Methods Mol.Biol.207:179-196(2008)，和/或解除抗原的残基，测试所得的变体VH或VL的结合亲和力。通过构建二级文库并自其进行再选择的亲和力成熟已被描述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，ed.,Human press,Totowa,NJ,(2001))中。在亲和力成熟的一些实施方案中，通过多种方法中任一种(例如，易错PCR、链改组或寡核苷酸定向诱变)将多样性引入选择用于成熟的可变基因种。然后产生二级 文库。然后筛选所述文库以鉴定任何具有所需亲和力的抗体变体。引入多样性的另一种方法包括CDR定向方法，其中若干CDR残基(例如，同时4-6个残基)被随机化。Alterations (eg, substitutions) can be made in the CDR regions, eg, to increase antibody affinity. Such alterations can be made in HVR "hotspots", i.e., codon-encoded residues that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008), and/or residues that relieve antigens, and the resulting variant VH or VL tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in HVR. Oogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genetic species selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Secondary levels are then generated library. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves a CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized.
在某些实施方案种，置换、插入或缺失可以发生在一个或多个CDR内，只要这样的改变不显著减小抗体结合抗原的能力。例如，不显著减小结合亲和力的保守改变(例如，本文中所述的保守修饰)可以在CDR中进行。此种改变可以例如在CDR中接触抗原的残基的外部。在上述提供的变体VH和VL序列的某些实施方案中，各CDR是未改变的，或含不超过一个、两个或三个氨基酸置换。In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such alterations do not significantly reduce the ability of the antibody to bind antigen. For example, conservative changes that do not significantly reduce binding affinity (eg, the conservative modifications described herein) can be made in the CDRs. Such alterations may be, for example, outside of the residues in the CDRs that contact the antigen. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.
术语“抗NKG2A抗体”、“结合NKG2A的抗体”、“NKG2A抗体”、“识别NKG2A的抗体”是指能够以足够的亲和力结合NKG2A的抗体，所述抗体可用作用于靶向NKG2A的诊断剂和/或治疗剂。在一个实施方案中，抗NKG2A抗体与不相关的、非NKG2A蛋白的结合程度小于所述抗体与NKG2A的约10％，如通过酶联免疫吸附实验(ELISA)测定。在某些实施方案中，抗NKG2A抗体结合NKG2A的表位，所述表位在来源于不同物种的NKG2A之间是保守的。The terms "anti-NKG2A antibody", "antibody that binds NKG2A", "NKG2A antibody", "antibody that recognizes NKG2A" refer to an antibody capable of binding NKG2A with sufficient affinity, which is useful as a diagnostic and/or therapeutic agent for targeting NKG2A. In one embodiment, the anti-NKG2A antibody binds to an irrelevant, non-NKG2A protein to an extent that is less than about 10% of the antibody's binding to NKG2A, as determined by an enzyme-linked immunosorbent assay (ELISA). In certain embodiments, an anti-NKG2A antibody binds an epitope of NKG2A that is conserved among NKG2A derived from different species.
在本文中，描述了具有基于Fab(抗原结合片段)的抗原结合区域的抗原结合蛋白，包括抗体Fab和抗体IgG4。其中使用NKG2A/CD94异源二聚体，从全人的天然的Fab噬菌体文库选择Fab。这些分子展示了精细的特异性。例如，该抗体仅识别NKG2A,不识别NKG2C、NKG2E。该抗体仅识别NKG2A/CD94异源二聚体，不识别NKG2C/CD94异源二聚体；该抗体仅识别过表达NKG2A/CD94异源二聚体的CHO-K1细胞，不识别过表达NKG2C/CD94、NKG2E/CD94以及CD94的CHO-K1细胞。本发明中如果没有特别说明，NKG2A指人的NKG2A。Herein, antigen-binding proteins having a Fab (fragment of antigen-binding)-based antigen-binding region are described, including antibody Fab and antibody IgG4. Here, the NKG2A/CD94 heterodimer was used to select Fab from a fully human native Fab phage library. These molecules exhibit fine specificity. For example, the antibody only recognizes NKG2A, and does not recognize NKG2C and NKG2E. The antibody only recognizes NKG2A/CD94 heterodimers, but not NKG2C/CD94 heterodimers; the antibody only recognizes CHO-K1 cells overexpressing NKG2A/CD94 heterodimers, and does not recognize CHO-K1 cells overexpressing NKG2C/CD94, NKG2E/CD94, and CD94. In the present invention, unless otherwise specified, NKG2A refers to human NKG2A.
在一些实施方案中，本发明包括具有Fab、IgG4序列的抗体，所述Fab序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。In some embodiments, the invention includes antibodies having a Fab, IgG4 sequence fused to one or more heavy chain constant regions to form an antibody with a human immunoglobulin Fc region to generate a bivalent protein, thereby increasing the overall affinity and stability of the antibody. Furthermore, the Fc moiety allows direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to antibodies for example in antigen quantification studies, for immobilization of antibodies for affinity measurements, for targeted delivery of therapeutics, testing of Fc-mediated cytotoxicity using immune effector cells, and many other applications.
本文提供的结果突出本发明抗体在靶向NKG2A时的特异性、灵敏性和效用。The results presented herein highlight the specificity, sensitivity and utility of the antibodies of the invention in targeting NKG2A.
本发明的抗体或抗体片段基于使用噬菌体展示鉴定和选择抗原结合片段(Fab)，所述抗原结合片段的氨基酸序列赋予抗体或抗体片段针对NKG2A的特异性并且形成本公开的全部抗原结合蛋白的基础。因此，所述Fab可以用来设计一系列不同“抗体或抗体片段”，包括例如全长抗体、其片段如F(ab’)2、融合蛋白、IgG4、多价抗体，即，具有针对相同抗原或不同抗原的多于一种特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(见Cuesta等人，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。Antibodies or antibody fragments of the invention are based on the use of phage display to identify and select antigen-binding fragments (Fabs) whose amino acid sequence confers specificity to the antibody or antibody fragment against NKG2A and forms the basis of all antigen-binding proteins of the disclosure. Thus, the Fab can be used to design a range of different "antibodies or antibody fragments" including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, IgG4, multivalent antibodies, i.e., antibodies with more than one specificity for the same antigen or different antigens, e.g., bispecific T cell-binding antibodies (BiTEs), tribodies, etc. (see Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotech Nology 28:355-362, 2010).
在具体实施例中，本发明提供全长抗体，其重链和轻链可以是全长(例如，抗体可以包括至少一条，优选两条完整重链，和至少一条，优选两条完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、 IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。In specific embodiments, the invention provides full-length antibodies, the heavy and light chains of which can be full-length (e.g., an antibody can include at least one, preferably two intact heavy chains, and at least one, preferably two intact light chains) or can include an antigen-binding portion (Fab, F(ab')2, Fv, or scFv). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE. The choice of antibody type will depend on the immune effector function the antibody is designed to elicit. In constructing recombinant immunoglobulins, the appropriate amino acid sequences for the constant regions of the various immunoglobulin isotypes and methods for producing the broad class of antibodies are known to those skilled in the art.
本发明提供了识别NKG2A的全人源抗体，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或DASSLES(SEQ ID NO:5)所示的LCDR2；和/或QQYDSYX1X2T所示的LCDR3，其中X1选自I、V、G、A、L、F或P，优选地，X1选自I或V；X2选自R、S、K、H、W、Y、C、M、N、Q或T，优选地，X2选自R或S。The present invention provides a fully human antibody that recognizes NKG2A, said antibody comprising a light chain variable region comprising LCDR1 represented by RASQSISSWLA (SEQ ID NO:4); and/or LCDR2 represented by DASSLES (SEQ ID NO:5); and/or LCDR3 represented by QQYDSYX 1 X 2 T, wherein X 1 is selected from I, V, G, A, L, F or P, preferably, X 1 is selected from I or V X2 is selected from R, S, K, H, W, Y, C, M, N, Q or T, preferably, X2 is selected from R or S.
本发明提供了识别NKG2A的全人源抗体，所述抗体包括重链可变区，所述重链可变区选自：The present invention provides a fully human antibody that recognizes NKG2A, the antibody includes a heavy chain variable region, and the heavy chain variable region is selected from:
(1)包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或GIIPIFGTAX1YAQKFQG(SEQ ID NO:130)所示的HCDR2，其中X1选自N、H、K、R、W、Y、S、C、M、Q或T，优选地，X1选自N或H；和/或GFDGMDY(SEQ ID NO:3)所示的HCDR3；或(1) comprising HCDR1 shown in SYAIS (SEQ ID NO: 1); and/or HCDR2 shown in GIIPIFGTAX 1 YAQKFQG (SEQ ID NO: 130), wherein X 1 is selected from N, H, K, R, W, Y, S, C, M, Q or T, preferably, X 1 is selected from N or H; and/or HCDR3 shown in GFDGMDY (SEQ ID NO: 3); or
(2)包含X1X2X3X4S(SEQ ID NO:131)所示的HCDR1；和/或AIX1X2X3X4GSTYYADSVKG(SEQ ID NO:132)所示的HCDR2；和/或GYDGFDY(SEQ ID NO:9)所示的HCDR3。其中HCDR1和HCDR2中X1X2X3X4位点的氨基酸选自下表2。(2) HCDR1 represented by X 1 X 2 X 3 X 4 S (SEQ ID NO: 131); and/or HCDR2 represented by AIX 1 X 2 X 3 X 4 GSTYYADSVKG (SEQ ID NO: 132); and/or HCDR3 represented by GYDGFDY (SEQ ID NO: 9). Wherein the amino acids at the X 1 X 2 X 3 X 4 position in HCDR1 and HCDR2 are selected from Table 2 below.
在一优选方案中，所述抗体还包括框架区，其中第1框架区的第30位氨基酸选自S、R、N、W、Y、C、M、Q、T、H、K、G、A、V、L、I、P、或F，优选地，选自S、R、N或G。In a preferred embodiment, the antibody further includes a framework region, wherein the 30th amino acid of the first framework region is selected from S, R, N, W, Y, C, M, Q, T, H, K, G, A, V, L, I, P, or F, preferably, selected from S, R, N or G.
表2.抗体HCDR1和HCDR2中X1X2X3X4位点的氨基酸
Table 2. Amino acids at X 1 X 2 X 3 X 4 positions in antibody HCDR1 and HCDR2
在一优选方案中，本发明提供了识别NKG2A的抗体，所述抗体包含重链可变区，所述重链可变区包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO:3或9 所示任一氨基酸序列的重链CDR3。在另一优选方案中，本发明提供了识别NKG2A的抗体包括：包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。在另一优选方案中，本发明提供了识别NKG2A的抗体包括：包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和/或包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3，和/或包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。优选地，所述识别NKG2A的抗体包括：包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3，和/或包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。更优选地，所述识别NKG2A的抗体包括：包含SEQ ID NO:1、7、12、14或16所示任一氨基酸序列的重链CDR1，和包含SEQ ID NO:2、8、11、13、15或17所示任一氨基酸序列的重链CDR2，和包含SEQ ID NO:3或9所示任一氨基酸序列的重链CDR3，和包含SEQ ID NO:4所示氨基酸序列的轻链CDR1，和包含SEQ ID NO:5所示氨基酸序列的轻链CDR2，和包含SEQ ID NO:6或10所示任一氨基酸序列的轻链CDR3。更优选地，所述识别NKG2A的抗体包括：包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或包含SEQ ID NO:7所示的HCDR1，SEQ ID NO:8所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:11所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或包含SEQ ID NO:12所示的HCDR1，SEQ ID NO:13所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或包含SEQ ID NO:14所示的HCDR1，SEQ ID NO:15所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或包含SEQ ID NO:16所示的HCDR1，SEQ ID NO:17所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3。In a preferred embodiment, the present invention provides an antibody that recognizes NKG2A, said antibody comprising a heavy chain variable region, said heavy chain variable region comprising heavy chain CDR1 of any amino acid sequence shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising heavy chain CDR2 of any amino acid sequence shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and/or comprising SEQ ID NO: 3 or 9 Heavy chain CDR3 of any amino acid sequence shown. In another preferred embodiment, the present invention provides an antibody recognizing NKG2A comprising: light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, and/or light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and/or light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10. In another preferred embodiment, the present invention provides an antibody recognizing NKG2A comprising: heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO:1, 7, 12, 14 or 16, and/or heavy chain CDR2 comprising any amino acid sequence shown in SEQ ID NO:2, 8, 11, 13, 15 or 17, and/or comprising heavy chain CDR3 of any amino acid sequence shown in SEQ ID NO:3 or 9, and/or comprising SEQ ID NO:4 The light chain CDR1 of the amino acid sequence, and/or the light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and/or the light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10. Preferably, the antibody that recognizes NKG2A includes: a heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO: 1, 7, 12, 14 or 16, and a heavy chain CDR2 comprising any amino acid sequence shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and a heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO: 3 or 9, and/or a light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 4, And light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10. More preferably, the antibody recognizing NKG2A comprises: heavy chain CDR1 comprising any amino acid sequence shown in SEQ ID NO: 1, 7, 12, 14 or 16, and heavy chain CDR2 comprising any amino acid sequence shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and heavy chain CDR3 comprising any amino acid sequence shown in SEQ ID NO: 3 or 9, and light chain CDR1 comprising an amino acid sequence shown in SEQ ID NO: 4, and Light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and light chain CDR3 comprising any amino acid sequence shown in SEQ ID NO:6 or 10. More preferably, the antibody that recognizes NKG2A includes: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6; or HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10; or HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:3; LCDR3 shown in Q ID NO:6; or comprising HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10; or HCDR1 shown in SEQ ID NO:14, HCDR shown in SEQ ID NO:15 2 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10; or HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:9; and LCDR3 shown in SEQ ID NO:10.
在另一方面，本发明提供了识别NKG2A的抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:18、22、26、28、30或32所示的氨基酸序列、或上述序列的变体。In another aspect, the present invention provides an antibody recognizing NKG2A, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or a variant of the above sequence.
在另一方面，本发明提供了识别NKG2A的抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:20或24所示的氨基酸序列、或上述序列的变体。 In another aspect, the present invention provides an antibody recognizing NKG2A, comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence.
考虑到这些重链可变区和轻链可变区序列各自可以结合NKG2A，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗NKG2A的结合分子。Given that each of these heavy and light chain variable region sequences can bind NKG2A, heavy and light chain variable region sequences can be "mixed and matched" to generate anti-NKG2A binding molecules of the invention.
在另一个方面，本发明提供了结合NKG2A的抗体的变体或其片段的变体。因而本发明提供了抗体或其片段，具有与重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛选、点突变等方法得到。如本申请实施例2采用的方法，以抗体A1和A2为母本抗体，采用噬菌体库筛选的方法进行突变改造。In another aspect, the invention provides variants of antibodies or fragments thereof that bind NKG2A. The invention thus provides antibodies or fragments thereof having heavy and/or light chain variable regions that are at least 80% identical to the variable region sequences of the heavy or light chains. Preferably, the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98%, most particularly 99%, including for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99% and 100%. Variants can be obtained by using the antibody described in the present application as the parent antibody, through yeast library screening, phage library screening, point mutation and other methods. As in the method adopted in Example 2 of the present application, antibody A1 and A2 were used as parental antibodies, and the method of phage library screening was used for mutation transformation.
在另一个方面，本发明提供了与前述的抗NKG2A的抗体识别相同的抗原决定部位的抗体。In another aspect, the present invention provides antibodies that recognize the same epitope as the aforementioned anti-NKG2A antibodies.
在另一个方面，本发明提供了与前述的抗NKG2A的抗体竞争性结合NKG2A的抗体。In another aspect, the present invention provides antibodies that compete for binding to NKG2A with the aforementioned anti-NKG2A antibodies.
在另一个方面，本发明提供了特异性结合NKG2A的抗体，所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或IgG4抗体。In another aspect, the invention provides an antibody that specifically binds NKG2A, said antibody being a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or IgG4 antibody.
在一优选方案中，以上所述的抗体不显著结合NKG2C、NKG2E或其组合。In a preferred embodiment, the antibodies described above do not significantly bind NKG2C, NKG2E or a combination thereof.
在一优选方案中，所述抗体结合NKG2A/CD94，不显著结合NKG2C/CD94、NKG2E/CD94或其组合；和/或，In a preferred embodiment, the antibody binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94 or a combination thereof; and/or,
所述抗体结合表达NKG2A/CD94的细胞，不显著结合表达NKG2C/CD94、NKG2E/CD94或其组合的细胞。The antibodies bind to cells expressing NKG2A/CD94 and do not significantly bind to cells expressing NKG2C/CD94, NKG2E/CD94, or combinations thereof.
抗体测定Antibody assay
通过本领域已知的多种测定可以鉴定、筛选本文中提供的抗NKG2A抗体或表征其物理/化学性质和/或生物学活性。包括例如ELISA、biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。Anti-NKG2A antibodies provided herein can be identified, screened for or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. Includes, for example, ELISA, biacore, Western blot and flow cytometry analysis. Suitable assays are described in detail in the Examples.
术语“亲和力”是指分子(例如：抗体)的单个结合位点及其结合配体(例如：抗原)之间非共价相互作用的力量总和。除非另外指出，如本文中使用的“结合亲和力”是指固有结合亲和力，其反应结合对的成员(例如：抗体和抗原)之间1：1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(Kd)代表。亲和力可以通过本领域中已知的常规方法测量，所述方法包括本文中所述的利用Biacore测定抗体的亲和力。本文中的抗体对NKG2A/CD94的“亲和力”以抗体的KD表示。抗体的KD是指抗体——抗原相互作用的平衡解离常数。抗体结合其抗原的KD值越大，其对所述具体抗原的结合亲和力越弱。The term "affinity" refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the determination of antibody affinity using Biacore as described herein. The "affinity" of an antibody for NKG2A/CD94 is expressed herein as the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The greater the KD value of an antibody for binding its antigen, the weaker its binding affinity for that particular antigen.
术语“EC50”，半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。The term "EC50", half maximum effect concentration (concentration for 50% of maximal effect, EC50) refers to the concentration that can cause 50% of the maximum effect.
抗原antigen
术语“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖 蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。The term "antigen" refers to a substance recognized and specifically bound by an antigen-binding unit. Antigens can include peptides, proteins, sugars Proteins, polysaccharides and lipids, fractions and combinations thereof. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
术语“表位”指可被抗体、B细胞、T细胞或工程化细胞识别的抗原或部分抗原。例如，表位可以是被抗体识别的肿瘤表位或病原体表位；抗体识别抗原内的多个表位。表位也可以突变。The term "epitope" refers to an antigen or part of an antigen that can be recognized by an antibody, B cell, T cell or engineered cell. For example, an epitope can be a tumor epitope or a pathogen epitope recognized by an antibody; an antibody recognizes multiple epitopes within an antigen. Epitopes can also be mutated.
术语“抗原决定部位”又称“抗原表位”或“表位”或“抗原决定簇”，包括任何能够被抗体结合的决定簇或区域。抗原表位是抗原中被靶向所述抗原的抗体结合的区域，包括与抗体直接接触的特定氨基酸。示例性，抗原表位可以由NKG2A蛋白序列的连续序列组成，也可以由NKG2A蛋白序列不连续的三维结构组成。示例性，本文中使用的抗原是NAG2A胞外区、NAG2C胞外区、NAG2E胞外区分别与CD94胞外区形成的NAG2A/CD94异源二聚体、NAG2C/CD94异源二聚体、NAG2E/CD94异源二聚体。The term "antigenic epitope", also known as "antigenic epitope" or "epitope" or "antigenic determinant", includes any determinant or region capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting the antigen, comprising specific amino acids that make direct contact with the antibody. Exemplarily, the antigenic epitope may consist of a continuous sequence of NKG2A protein sequence, or may consist of a discontinuous three-dimensional structure of NKG2A protein sequence. Exemplarily, the antigens used herein are NAG2A/CD94 heterodimer, NAG2C/CD94 heterodimer, NAG2E/CD94 heterodimer formed by NAG2A extracellular region, NAG2C extracellular region, NAG2E extracellular region and CD94 extracellular region respectively.
免疫缀合物Immunoconjugate
本发明还提供了免疫缀合物，所述的免疫辍合物包括本文所述的抗体，以及与之连接的功能性分子。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成缀合物。The present invention also provides immunoconjugates, which include the antibodies described herein, and functional molecules linked thereto. The antibody and the functional molecule can form a conjugate through covalent connection, coupling, attachment, cross-linking and the like.
术语“连接”或“融合”在本文中可互换使用。这些术语是指通过包括化学缀合或重组方法的任何手段将两个以上化学元件或组件连接在一起。“框内融合”是指以维持原始开放阅读框(ORF)的正确阅读框的方式连接两个或更多个ORF以形式连续的较长的ORF。因此，所得到的重组融合蛋白是含有两个或更多个片段的单一蛋白质，这些片段对应于由原始ORF编码的多肽(这些片段在自然状态通常不是如此连接)。尽管阅读框因此在整个融合片段中是连续的，但这些片段可以在物理上或空间上通过例如框内连接序列(例如“flexon”)分开。The terms "linked" or "fused" are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or modules by any means including chemical conjugation or recombinant methods. "In-frame fusion" refers to the joining of two or more ORFs to form a contiguous longer ORF in a manner that maintains the correct reading frame of the original open reading frame (ORF). Thus, the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (the segments are not normally so linked in nature). Although the reading frames are thus contiguous throughout the fusion fragments, the fragments may be separated physically or spatially by, for example, in-frame linking sequences (eg, "flexons").
本发明另一方面提供了编码本发明的至少一种抗体、其功能变体或免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
嵌合受体chimeric receptor
术语“嵌合受体”，即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。The term "chimeric receptor" refers to a fusion molecule formed by linking DNA fragments from different sources or corresponding cDNAs of proteins by genetic recombination technology, including extracellular domains, transmembrane domains and intracellular domains. Chimeric receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor, T Cell Antigen Coupler (TAC).
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结 构域。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域，在一个方面，刺激性分子为与T细胞受体复合体结合的δ链；在一个方面，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。The term "chimeric antigen receptor" (CAR) includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling junction. domain. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and/or a co-stimulatory molecule. In one aspect, the stimulatory molecule is a delta chain bound to a T cell receptor complex; in one aspect, the cytoplasmic signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB (ie, CD137), CD27 and/or CD28.
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该嵌合T细胞受体整合进T细胞上表达的TCR。The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides constituting TCR, which can bind to surface antigens on target cells and interact with other polypeptides of the complete TCR complex, usually colocalized on the surface of T cells. The chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the stimulation domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit is operatively linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is integrated into T cells for expression TCR.
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。The term "T cell antigen coupler (T cell antigen coupler, TAC)" includes three functional domains: 1. Antigen binding domain, including single-chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting groups; 2. Extracellular domain, single-chain antibody that binds to CD3, so that TAC receptor and TCR receptor are close; 3. Transmembrane domain and intracellular domain of CD4 co-receptor, Among them, the intracellular domain-linked protein kinase LCK catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
术语“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。胞内信号传导结构域可以包括分子的全部细胞内部分、或全部天然胞内信号传导结构域、或其功能片段或衍生物。The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate the activity of the cell via defined signaling pathways by producing secondary messengers or by acting as effectors in response to such messengers. The intracellular signaling domain may include the entire intracellular portion of the molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.
术语“初级信号域”以刺激性方式调节TCR复合物的初始活化。一方面，初级信号域由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号域可以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本发明中尤其有用的包含ITAM的初级信号域的例子包括但不限于，源自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d的序列，在特例的本发明CAR中，在任何一个或多个本发明CAR中胞内信号传导结构域包含胞内信号传导序列，例如CD3δ的初级信号域。The term "primary signaling domain" regulates the initial activation of the TCR complex in a stimulatory manner. In one aspect, the primary signaling domain is elicited by, for example, the binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating a T cell response (including, but not limited to, proliferation, activation, differentiation, etc.). Primary signaling domains acting in a stimulatory manner may comprise immunoreceptor tyrosine activation motifs or signaling motifs of ITAMs. Examples of ITAM-containing primary signaling domains that are particularly useful in the present invention include, but are not limited to, sequences derived from TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d. primary signal domain.
术语“共刺激信号域”指“共刺激分子”，指与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号。为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，例如，但不限于，增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于，MHC I类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。 The term "co-stimulatory signal domain" refers to "co-stimulatory molecule", which refers to a signal that combines with a cell-stimulatory signal molecule, such as TCR/CD3, to result in T cell proliferation and/or up-regulation or down-regulation of key molecules. is a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137).
在本发明中，一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自刺激分子的功能信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自共刺激分子的功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域和胞内传导结构域，所述胞内信号传导结构域包含源自一个或多个共刺激分子的至少两个功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR在CAR融合蛋白的氨基酸(ND端)包含可选的前导序列。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。In the present invention, in one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino acid (ND terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during the cellular processing and localization of the CAR to the cell membrane.
术语“CD3δ(也称为CD3 Zeta)”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3δ结构域”定义为来自ξ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，ξ的胞质结构域包含GenBan登录号BAG36664.1的残基52至164、其功能性直向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。The term "CD3 delta (also known as CD3 Zeta)" is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like. "CD3 delta domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic domain of ξ comprises residues 52 to 164 of GenBan Accession No. BAG36664.1, a functional ortholog thereof - equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like.
在一些实施方案中，本发明嵌合受体是嵌合抗原受体。In some embodiments, the chimeric receptors of the invention are chimeric antigen receptors.
本发明提供了嵌合抗原受体(Chimeric Antigen Receptor，CAR)，其包含本文所述的胞外结合结构域、跨膜域和胞内域。常见地，CAR的胞外结合结构域(或称为结构区)来源于小鼠或人源化或人的单克隆抗体。The present invention provides a chimeric antigen receptor (Chimeric Antigen Receptor, CAR), which comprises the extracellular binding domain, transmembrane domain and intracellular domain described herein. Commonly, the extracellular binding domain (or called structural region) of CAR is derived from mouse or humanized or human monoclonal antibody.
嵌合抗原受体通常包含胞外抗原结合区或者抗体。在一些实施例中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区可以被人源化。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施例中，所述胞外抗原结合区可以是非人的。Chimeric antigen receptors typically comprise an extracellular antigen-binding domain or an antibody. In some embodiments, the extracellular antigen binding region can be fully human. In other cases, the extracellular antigen binding domain can be humanized. In other cases, the extracellular antigen binding region may be of murine origin, or a chimera in said extracellular antigen binding region may consist of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.
嵌合抗原受体可以设计多种抗原结合区，包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然配体。在一些实施例中，胞外抗原结合区可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合 区。Chimeric antigen receptors can be designed with a variety of antigen-binding domains, including single-chain variable fragments (scFv) derived from antibodies, fragment antigen-binding domains (Fab) selected from libraries, single-domain fragments, or natural ligands that engage their cognate receptors. In some embodiments, the extracellular antigen binding region may comprise scFv, Fab or natural ligand, and any derivatives thereof. An extracellular antigen binding region can refer to a molecule other than an intact antibody, which can comprise a portion of an intact antibody and can bind to the antigen to which the intact antibody binds. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies, linear antibodies; single chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. An extracellular antigen-binding region, such as a scFv, Fab, or natural ligand, can be part of a CAR that determines antigen specificity. The extracellular antigen binding domain can bind any complementary target. Extracellular antigen binding regions can be derived from antibodies of known variable region sequences. Extracellular antigen binding regions can be derived from antibody sequences obtained from available mouse hybridomas. Alternatively, extracellular antigen binding can be obtained from total excision sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TILs). district.
在一些实施例中，CAR的胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。In some embodiments, the binding specificity of the extracellular antigen-binding region of the CAR can be determined by complementarity determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity can be determined by the light and heavy chain CDRs.
在一些实施例中，CAR的胞外抗原结合区包括铰链或间隔区。术语铰链和间隔区可以互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施例中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于胞外抗原结合区靶向靶上的表位的位置。在一些实施例中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。所述半胱氨酸和脯氨酸残基可影响所述CAR的性能。可以根据所使用的胞外抗原结合区来调节铰链。铰链可以是任何长度的。In some embodiments, the extracellular antigen binding region of the CAR includes a hinge or spacer. The terms hinge and spacer are used interchangeably. The hinge can be considered as part of the CAR used to provide flexibility to the extracellular antigen-binding region. In some embodiments, the hinge can be used to detect a CAR on the cell surface of a cell, particularly when antibodies to detect the extracellular antigen binding region are not functional or available. For example, the length of a hinge derived from an immunoglobulin may need to be optimized, depending on where the epitope on the target is targeted by the extracellular antigen binding region. In some embodiments, the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of the CD8α molecule. The CD8α hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 coreceptors and MHC molecules. The cysteine and proline residues can affect the performance of the CAR. The hinge can be adjusted depending on the extracellular antigen binding region used. Hinges can be of any length.
CAR的跨膜结构域(或称为结构区)可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施例中，跨膜部分可以包含CD8α区。The transmembrane domain (or structural region) of CAR can anchor the CAR on the plasma membrane of the cell. The native transmembrane portion of CD28 can be used in CAR. In other cases, it is also possible to use the native transmembrane portion of CD8α in the CAR. "CD8" may be a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity. A "CD8 nucleic acid molecule" may be a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane region may be the natural transmembrane part of CD28, and "CD28" may refer to a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with NCBI reference number: NP_006130 or a fragment thereof having stimulating activity. A "CD28 nucleic acid molecule" may be a polynucleotide encoding a CD28 polypeptide. In some embodiments, the transmembrane portion may comprise a CD8α region.
CAR的(细)胞内信号传导区可以负责活化包含所述CAR的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌，如IL-2，TNF-α,γ-IFN等。因此，术语细胞内信号传导区是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区，但是在许多情况下，不必使用信号结构域的整个链。在一些实施例中，使用细胞内信号传导区的截短部分。在一些实施例中，术语细胞内信号传导区因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。The (cellular) signaling region of the CAR may be responsible for activating at least one of the effector functions of an immune response cell comprising said CAR. CAR can induce the effector function of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines, such as IL-2, TNF-α, γ-IFN, etc. Thus, the term intracellular signaling region refers to the portion of a protein that transduces the signal for effector functions and directs the cell to specific functions. While usually the entire intracellular signaling region can be used, in many cases it is not necessary to use the entire chain of signaling domains. In some embodiments, truncated portions of intracellular signaling regions are used. In some embodiments, the term intracellular signaling region is thus intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
在CAR中使用的信号结构域(或称为结构区)的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。A preferred example of the signaling domain (or called structural region) used in CAR may include the cytoplasmic sequence of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction after target-receptor binding, and any derivative or variant sequences thereof and any synthetic sequences of these sequences with the same functionality.
在一些实施例中，所述CAR的细胞内信号传导区可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自TCRδ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。然而， 在优选的实施例中，细胞内信号结构域衍生自CD3δ链。In some embodiments, the intracellular signaling region of the CAR may contain a known signaling motif of an immunoreceptor tyrosine activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCRδ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. However, In preferred embodiments, the intracellular signaling domain is derived from the CD3 delta chain.
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3δ结构域，也称为T细胞受体CD3δ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3δ主要是指人类CD3δ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，不需要全T细胞受体CD3δ链，并且其包含T细胞受体CD3δ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。An example of a T cell signaling domain containing one or more ITAM motifs is the CD3 delta domain, also known as the T cell receptor CD3 delta chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition of several intracellular signal transduction pathways with activation of main effectors of T cells. As used herein, CD3delta primarily refers to human CD3delta and its isoforms, as known from Swissprot entry P20963, including proteins with substantially identical sequences. As part of a chimeric antigen receptor, the full T cell receptor CD3 delta chain is not required and any derivative thereof comprising the signaling domain of the T cell receptor CD3 delta chain is suitable, including any functional equivalents thereof.
细胞内信号传导结构域(或称为结构区)可以选自表1的任何一个共刺激结构域。在一些实施例中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或至多约100％的同一性。The intracellular signal transduction domain (or called structural region) can be selected from any costimulatory domain in Table 1. In some embodiments, a domain can be modified such that the identity to a reference domain can be from about 50% to about 100%. Any one of the domains of Table 1 can be modified such that the modified form can comprise about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% identity.
CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如δ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。The intracellular signaling region of the CAR may further comprise one or more co-stimulatory domains. The intracellular signaling domain may comprise a single co-stimulatory domain, such as the delta chain (first generation CAR) or its combination with CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling domain may comprise two co-stimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。Together with intracellular signaling domains such as CD8, these co-stimulatory domains can generate downstream activation of kinase pathways that support gene transcription and functional cellular responses. The costimulatory domain of CAR can activate proximal signaling proteins associated with CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB/OX40 (TNF-receptor-associated factor adapter protein) pathways, as well as MAPK and Akt activation.
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。在本领域众所周知的，在幼稚T细胞中，T细胞受体的单独结合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。In some cases, signals generated by CARs may be combined with auxiliary or co-stimulatory signals. For co-stimulatory signaling domains, chimeric antigen receptor-like complexes can be designed to contain several possible co-stimulatory signaling domains. It is well known in the art that in naive T cells, binding of the T cell receptor alone is not sufficient to induce full activation of the T cell into a cytotoxic T cell. A second co-stimulatory signal is required for full productive T cell activation. Several receptors have been reported to provide co-stimulation to T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4-1BB. The signaling pathways used by these co-stimulatory molecules all work synergistically with primary T cell receptor activation signals. The signals provided by these co-stimulatory signaling domains can cooperate with main effector activation signals derived from one or more ITAM motifs (such as CD3zeta signaling domains) and can fulfill the requirements of T cell activation.
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一些实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。In some embodiments, adding costimulatory domains to chimeric antigen receptor-like complexes can enhance the efficacy and durability of engineered cells. In other embodiments, the T cell signaling domain and the co-stimulatory domain are fused to each other to constitute the signaling domain.
表3.共刺激结构域

Table 3. Co-stimulatory domains

含有抗NKG2A抗体的嵌合抗原受体Chimeric antigen receptor containing anti-NKG2A antibody
本发明还提供多种嵌合抗原受体(CAR)，其中包含本发明的抗体或其片段，该CAR-T细胞表现出抗肿瘤性质。一些实施案例中，用编码CAR的病毒载体转导细胞(例如T细胞)。在一些实施案例中，病毒载体是慢病毒载体。一些实施方案中，细胞可以稳定地表达CAR。The present invention also provides various chimeric antigen receptors (CARs) comprising the antibodies or fragments thereof of the present invention, and the CAR-T cells exhibit anti-tumor properties. In some embodiments, cells (eg, T cells) are transduced with a CAR-encoding viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express the CAR.
在一优选例中，CAR的NKG2A结合部分是scFv，与其所来自的Fab抗体相比，保持等价的亲和结合力，例如其以相当的功效结合相同抗原。该抗体片段是功能性的，由此其提供生物化学反应，例如激活免疫反应、抑制从其靶抗原的信号传导起始、抑制激酶活性等。In a preferred example, the NKG2A binding portion of the CAR is scFv, which maintains equivalent affinity and binding capacity compared with the Fab antibody from which it is derived, for example, it binds the same antigen with comparable efficacy. The antibody fragment is functional in that it provides a biochemical response, such as activation of an immune response, inhibition of signaling initiation from its target antigen, inhibition of kinase activity, and the like.
一方面，CAR的抗NKG2A抗原结合结构域是全人源抗体片段。In one aspect, the anti-NKG2A antigen-binding domain of the CAR is a fully human antibody fragment.
一方面，本发明CAR将特定抗体的抗原结合结构域和胞内信号传导分子组合在一起。例如，一些方面，胞内信号传导分子包括但不限于，CD3δ、4-1BB和CD28信号传导模块及其组合。 In one aspect, the CAR of the present invention combines the antigen-binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, intracellular signaling molecules include, but are not limited to, CD3delta, 4-1BB, and CD28 signaling modules and combinations thereof.
一方面，NKG2A-CAR包含至少一个胞内信号传导结构域，其选择CD137(4-1BB)信号传导结构域、CD28信号传导结构域、CD3δ信号结构域，及其任何组合。一方面，NKG2A-CAR包含至少一个胞内信号传导结构域，其来自一个或多个非CD137(4-1BB)或CD28的共刺激分子。In one aspect, the NKG2A-CAR comprises at least one intracellular signaling domain selected from the group consisting of CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3δ signaling domain, and any combination thereof. In one aspect, the NKG2A-CAR comprises at least one intracellular signaling domain derived from one or more co-stimulatory molecules other than CD137(4-1BB) or CD28.
作为示例性的，NKG2A-CAR的序列可以是：As an example, the sequence of NKG2A-CAR can be:
嵌合抗原受体一，具有SEQ ID NO:64所示的胞外域、SEQ ID NO：95所示的铰链域、SEQID NO：97所示的跨膜域、SEQ ID NO：101所示的共刺激信号域、以及SEQ ID NO：105所示的初级信号域(A4-BBZ)；或A chimeric antigen receptor having an extracellular domain set forth in SEQ ID NO:64, a hinge domain set forth in SEQ ID NO:95, a transmembrane domain set forth in SEQ ID NO:97, a co-stimulatory signaling domain set forth in SEQ ID NO:101, and a primary signaling domain (A4-BBZ) set forth in SEQ ID NO:105; or
嵌合抗原受体二，具有SEQ ID NO:66所示的胞外域、SEQ ID NO：95所示的铰链域、SEQID NO：97所示的跨膜域、SEQ ID NO：101所示的共刺激信号域、以及SEQ ID NO：105所示的初级信号域(A5-BBZ)。Chimeric antigen receptor two has an extracellular domain shown in SEQ ID NO:66, a hinge domain shown in SEQ ID NO:95, a transmembrane domain shown in SEQ ID NO:97, a co-stimulatory signal domain shown in SEQ ID NO:101, and a primary signal domain (A5-BBZ) shown in SEQ ID NO:105.
示例性的，嵌合受体的氨基酸序列如SEQ ID NO：115或116所示。Exemplarily, the amino acid sequence of the chimeric receptor is shown in SEQ ID NO: 115 or 116.
上述嵌合抗原受体的跨膜域和胞内域，本领域技术人员可以选择常规的跨膜域和胞内域进行替换，且均落入本申请的保护范围。The transmembrane domain and intracellular domain of the chimeric antigen receptor above can be replaced by conventional transmembrane domains and intracellular domains by those skilled in the art, and all of them fall within the protection scope of the present application.
核酸、载体、病毒、宿主细胞Nucleic acid, vector, virus, host cell
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。The terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer to the sequence or sequence of deoxyribonucleotides along a deoxyribose nucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.
当用于指核苷酸序列时，本文所用的术语“序列”包括DNA或RNA，并且可以是单链或双链。As used herein, the term "sequence" when used to refer to a nucleotide sequence includes DNA or RNA, and may be single- or double-stranded.
术语“靶序列”是指与指导序列具有互补性的序列，靶序列与指导序列之间互补配对促进CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。The term "target sequence" refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.
术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。The term sequence "identity" determines the percent identity by comparing two best-matched sequences over a comparison window (e.g., at least 20 positions), wherein portions of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions (i.e., gaps), e.g., a gap of 20% or less (e.g., 5 to 15%, or 10 to 12%) for the two best-matched sequences compared to a reference sequence (which does not contain additions or deletions). Percentages are typically calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to yield the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to yield the percent sequence identity.
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸 序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，如质粒、病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising a An expression control sequence operably linked to the sequence. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or by an in vitro expression system. Expression vectors include all those known in the art, such as plasmids, viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。The term "vector" as used herein is a composition comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, and the like.
本文使用的术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。The term "lentivirus" as used herein refers to a genus of the Retroviridae family. Retroviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the host cell's DNA, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide the means to achieve significant levels of gene transfer in vivo.
术语“内源”是指一个核酸分子或多肽等来自生物体自身。The term "endogenous" means that a nucleic acid molecule or polypeptide etc. is derived from the organism itself.
本文所用的术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。The term "exogenous" as used herein refers to a function of a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not expressed endogenously in the organism itself, or the expression level is insufficient to achieve overexpression.
本文所用的术语“外源蛋白”可以是识别靶抗原的外源转入细胞的蛋白，如外源受体(即本文中前述“嵌合受体”)。The term "exogenous protein" used herein may be a protein introduced into cells exogenously that recognizes a target antigen, such as an exogenous receptor (ie, the aforementioned "chimeric receptor" herein).
本文中使用的术语“宿主”是指接受移植物移植的受体，在一些实施方式中，可以是接受外源细胞植入的个体，如人。The term "host" as used herein refers to a recipient receiving a transplant, and in some embodiments, may be an individual, such as a human, receiving exogenous cells implanted.
术语“分离的”是指与细胞成分或其他成分相分离，在这些成分中，多核苷酸、肽、多肽、蛋白质、抗体或其片段在自然状态通常是相关联的。如本领域技术人员将理解，非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”以将其与天然存在的对应物区分开。此外，“浓缩”、“分离”或“稀释”的多核苷酸、肽、多肽、蛋白质、抗体或其片段可与其天然存在的对应物区分开，因为每体积分子的浓度或数量大于(“浓缩”)或小于(“稀释”)其天然存在的配对物的浓度。富集程度可以以绝对基础测量，例如每溶液体积的重量，或者可以相对于存在于源混合物中的另一潜在的干扰物测量。在一些实施方案中，本发明的技术方案优选的富集程度更高。因此，例如，优选2倍富集、更优选10倍富集、更优选100倍富集、更优选1000倍富集。也可以通过人工组装的方法，例如通过化学合成或重组表达，从而提供“分离的”物质。The term "isolated" means separated from cellular components or other components in which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are normally associated in their natural state. As will be appreciated by those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not need to be "isolated" to distinguish it from its naturally occurring counterpart. Furthermore, a "concentrated", "isolated" or "diluted" polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is distinguishable from its naturally occurring counterpart because the concentration or number of molecules per volume is greater ("concentrated") or less than ("diluted") the concentration of its naturally occurring counterpart. The degree of enrichment can be measured on an absolute basis, such as weight per solution volume, or can be measured relative to another potential interferent present in the source mixture. In some embodiments, the preferred enrichment degree of the technical solution of the present invention is higher. Thus, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, 1000-fold enrichment is more preferred. An "isolated" substance can also be provided by artificial means of assembly, for example, by chemical synthesis or recombinant expression.
本发明提供了编码识别NKG2A的抗体或其片段分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。The present invention provides an isolated nucleic acid encoding an NKG2A-recognizing antibody or a fragment thereof, a vector, and a host cell comprising the nucleic acid or vector. Nucleic acids can be located in intact cells, in cell lysates, or in partially or substantially purified form.
可以使用标准的分子生物学技术获得本发明的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变 化。Nucleic acids of the invention can be obtained using standard molecular biology techniques, for example, cDNAs encoding antibody light and heavy chains or encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Methods for introducing exogenous nucleic acids into host cells are generally known in the art and may vary with the host cell used. change.
优选的，本发明核酸分子是选自编码重链可变区的SEQ ID NO:19、23、27、29、31或33，和/或选自编码轻链可变区的SEQ ID NO:21或25。更优选的，是这样的核酸分子，所述核酸分子包含SEQ ID NO:19的重链可变区序列，和包含SEQ ID NO:21的轻链可变区序列；或者包含SEQ ID NO:23的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列；或者包含SEQ ID NO:27的重链可变区序列，和包含SEQ ID NO:21的轻链可变区序列；或者包含SEQ ID NO:29的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列；或者包含SEQ ID NO:31的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列；或者包含SEQ ID NO:33的重链可变区序列，和包含SEQ ID NO:25的轻链可变区序列。Preferably, the nucleic acid molecule of the present invention is selected from SEQ ID NO: 19, 23, 27, 29, 31 or 33 encoding the variable region of the heavy chain, and/or selected from SEQ ID NO: 21 or 25 encoding the variable region of the light chain. More preferably, it is a nucleic acid molecule comprising a heavy chain variable region sequence of SEQ ID NO: 19, and a light chain variable region sequence comprising SEQ ID NO: 21; or a heavy chain variable region sequence comprising SEQ ID NO: 23, and a light chain variable region sequence comprising SEQ ID NO: 25; or a heavy chain variable region sequence comprising SEQ ID NO: 27, and a light chain variable region sequence comprising SEQ ID NO: 21; or A heavy chain variable region sequence comprising SEQ ID NO:29, and a light chain variable region sequence comprising SEQ ID NO:25; or a heavy chain variable region sequence comprising SEQ ID NO:31, and a light chain variable region sequence comprising SEQ ID NO:25; or a heavy chain variable region sequence comprising SEQ ID NO:33, and a light chain variable region sequence comprising SEQ ID NO:25.
在一个实施方案中，提供一种或多种包含上述核酸的载体(例如，表达载体)。In one embodiment, one or more vectors (eg, expression vectors) comprising the nucleic acids described above are provided.
术语“细胞”指人或非人动物来源的细胞。The term "cell" refers to a cell of human or non-human animal origin.
术语“宿主细胞”指被引入外源核酸的细胞，包括此种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括转化的原代细胞及来源于其的后代(不考虑传代次数)。后代的核酸内容可以与亲本细胞不完全相同，并且可以含有突变。本文中包括具有与对于原始转化的细胞中筛选或选择的相同的功能或生物学活性的突变体后代。The term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include transformed primary cells and progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be identical to that of the parental cells and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
术语“NKG2A或NKG2A/CD94阳性宿主细胞”是指在细胞表面上表达NKG2A/CD94的宿主细胞，这些细胞可以通过例如使用抗体的流式细胞术来检测，这些抗体特异性识别CD94和NKG2A上的组合表位或单独NKG2A上的表位。The term "NKG2A or NKG2A/CD94 positive host cell" refers to a host cell expressing NKG2A/CD94 on the cell surface, which can be detected, for example, by flow cytometry using antibodies that specifically recognize a combined epitope on CD94 and NKG2A or an epitope on NKG2A alone.
在一些实施方案中，所述宿主细胞是免疫效应细胞。In some embodiments, the host cells are immune effector cells.
术语“免疫效应细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。“免疫效应功能或免疫效应应答”是指免疫效应细胞，例如增强或促进靶细胞的免疫攻击的功能或反应。例如，免疫效应功能或应答是指促进靶细胞的杀伤或者抑制生长或增殖的T细胞或NK细胞的属性。The term "immune effector cells" refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, etc. In some embodiments, the immune effector cells are T cells, NK cells, NKT cells. In some embodiments, the T cells may be autologous T cells, allogeneic T cells, allogeneic T cells. In some embodiments, the NK cells may be allogeneic NK cells. "Immune effector function or immune effector response" refers to immune effector cells, eg, functions or responses that enhance or facilitate immune attack of target cells. For example, immune effector function or response refers to the properties of T cells or NK cells that promote killing of target cells or inhibit growth or proliferation.
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。The term "artificially engineered cell with immune effector cell function" refers to a cell or cell line that does not have immune effector after being artificially modified or stimulated by a stimulant, the cell acquires immune effector cell function. For example, 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情 况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。In some instances, "T cells" may be pluripotent stem cells derived from bone marrow that differentiate and mature into immunocompetent mature T cells within the thymus. In some cases, "T cells" may be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as "T cells" may be cells comprising at least one T cell subset: memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells (Tem). in some situations In some cases, "T cells" can be a specific subtype of T cells, such as γδ T cells.
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。在一个实施方案中，可以从健康供体，或来自诊断患有肿瘤的患者衍生细胞获得T细胞。T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, T cells can be obtained from blood collected from an individual using any number of techniques known to those of skill in the art, such as Ficoll(TM) isolation. In one embodiment, the cells from the circulating blood of the individual are obtained by apheresis. Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps. In one embodiment, T cells can be obtained from a healthy donor, or from cells derived from a patient diagnosed with a tumor.
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。The term "peripheral blood mononuclear cell" (peripheral blood mononuclear cell, PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes and the like.
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指NK细胞、T细胞逐步活化的过程。The terms "activation" and "activation" are used interchangeably and can refer to the process by which a cell transitions from a quiescent state to an active state. The process can include responses to antigenic, phenotypic or genetic changes in migration and/or functional activity status. For example, the term "activation" may refer to the process of gradual activation of NK cells and T cells.
术语“T细胞活化”或“T细胞激活”指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。The term "T cell activation" or "T cell activation" refers to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
在另一实施方案中，提供包含上述核酸的宿主细胞。宿主细胞包含(例如，转导有)：(1)载体，所述载体包含核酸，所述核酸编码包含抗体VL的氨基酸序列和包含抗体VH的氨基酸序列，或(2)包含编码包含抗体VL的氨基酸序列的核酸的第一载体，和包含编码包含抗体VH的氨基酸序列的核酸的第二载体。在一个实施方案中，宿主细胞是真核的，例如，中国仓鼠卵巢(CHO)细胞或淋巴细胞(例如，YO、NSO、Sp20细胞)。In another embodiment, a host cell comprising the nucleic acid described above is provided. The host cell comprises (e.g., is transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, eg, Chinese Hamster Ovary (CHO) cells or lymphocytes (eg, YO, NSO, Sp20 cells).
在另一实施方案中，所述宿主细胞表达本发明所述的嵌合受体。In another embodiment, the host cell expresses a chimeric receptor of the invention.
在另一实施方案中，所述宿主细胞包括T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、和/或干细胞衍生的免疫效应细胞。In another embodiment, the host cells comprise T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, and/or stem cell-derived immune effector cells.
在另一实施方案中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；优选地，所述T细胞为自体/同种异体T细胞；优选地，所述T细胞为原代T细胞；优选地，所述T细胞来源于人的自体T细胞。In another embodiment, the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells; preferably, the T cells are autologous/allogeneic T cells; preferably, the T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
在另一实施方案中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。In another embodiment, the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), γδT cells or combinations thereof.
在另一实施方案中，所述宿主细胞结合表达NKG2A/CD94的细胞，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。In another embodiment, the host cell binds to cells expressing NKG2A/CD94 and does not significantly bind NKG2C/CD94, NKG2E/CD94, or a combination thereof.
在另一实施方案中，所述宿主细胞还携带外源的细胞因子的编码序列。In another embodiment, the host cell also carries coding sequences for exogenous cytokines.
在另一实施方案中，所述宿主细胞还可以表达除了上述结合抗原的受体以外的另一种嵌合抗原受体。 In another embodiment, the host cell may also express another chimeric antigen receptor in addition to the antigen-binding receptors described above.
在另一实施方案中，所述宿主细胞还可以表达趋化因子受体。In another embodiment, the host cell may also express chemokine receptors.
在另一实施方案中，所述宿主细胞还可以表达安全开关。In another embodiment, the host cell can also express a safety switch.
在另一实施方案中，所述宿主细胞能杀伤活化的NK细胞。In another embodiment, the host cell is capable of killing activated NK cells.
在一个实施例中，提供制备抗NKG2A抗体的方法，其中所述方法包括在适合于表达如上所述的抗体的条件下培养包含编码所述抗体的核酸的宿主细胞，和任选地从宿主细胞(或宿主细胞培养基)回收抗体。In one embodiment, a method of making an anti-NKG2A antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).
为了表达蛋白质，可以将编码本发明抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。用于本发明的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本发明中来表达抗体。In order to express the protein, the nucleic acid encoding the antibody of the present invention can be incorporated into an expression vector. A variety of expression vectors are available for protein expression. Expression vectors may include self-replicating extrachromosomal vectors, or vectors that integrate into the host genome. Expression vectors useful in the present invention include, but are not limited to, those that enable protein expression in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. A variety of expression vectors are available commercially or otherwise, as known in the art. Antibodies may be expressed in the present invention.
在一优选例中，所述宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；和/或所述宿主细胞与改善其相关的一种或多种副作用的药剂联合施用；和/或所述宿主细胞与表达靶向NKG2A之外的嵌合抗原受体的宿主细胞联合施用。In a preferred example, the host cell is administered in combination with a drug that enhances its function, preferably, a chemotherapy drug; and/or the host cell is administered in combination with a drug that improves one or more side effects associated therewith; and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting other than NKG2A.
基因编辑的细胞gene edited cells
在一实例中，采用包含本发明提供的gRNA的CRISPR系统敲除细胞内源性TCR、B2M、NKG2A和/或CIITA。对细胞(例如，T细胞或NKT细胞)的遗传修饰可以通过用重组核酸分子转导基本上均质的细胞群来完成。In one example, endogenous TCR, B2M, NKG2A and/or CIITA in cells are knocked out using a CRISPR system comprising gRNA provided by the present invention. Genetic modification of cells (eg, T cells or NKT cells) can be accomplished by transducing a substantially homogeneous population of cells with a recombinant nucleic acid molecule.
在一实例中，细胞内源性TCR、B2M、NKG2A和/或CIITA基因敲除后，细胞中TCR、B2M、NKG2A和/或HLA-II低表达或不表达。TCR、B2M、NKG2A和/或HLA-II低表达或不表达是指细胞中CR、B2M、NKG2A和/或HLA-II的表达减少至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。更具体而言，CR、B2M、NKG2A和/或HLA-II低表达或不表达分别是指细胞中CR、B2M、NKG2A和/或HLA-II的含量降低至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。可以通过本领域内已知的任何合适的方法，如ELISA、免疫组织化学、免疫印迹(Western Blotting)或流式细胞术使用CR、B2M、NKG2A和/或HLA-II的特异性抗体测定细胞中蛋白的表达或含量。In one example, after the endogenous TCR, B2M, NKG2A and/or CIITA genes in the cells are knocked out, TCR, B2M, NKG2A and/or HLA-II are expressed low or not in the cells. Low or no expression of TCR, B2M, NKG2A and/or HLA-II means that the expression of CR, B2M, NKG2A and/or HLA-II in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. More specifically, low or no expression of CR, B2M, NKG2A and/or HLA-II means that the content of CR, B2M, NKG2A and/or HLA-II in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. The expression or content of proteins in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies to CR, B2M, NKG2A and/or HLA-II.
由于供体和受体(或称为宿主)之间的免疫遗传学差异，在进行外源供体移植时，作为外源移植物的供体会受到宿主体内的免疫细胞(例如NK细胞)识别和攻击，进而抑制或者清除供体，产生宿主抗移植物反应(HVGR)。如在异体细胞移植中，当异体细胞的HLA-I类分子的缺失，可以降低宿主CD8+介导的细胞免疫排斥作用。在一实例中，本发明提供内源性HLA-II/B2M低表达或不表达的免疫细胞。Due to the immunogenetic differences between the donor and the recipient (or host), during exogenous donor transplantation, the donor as an exogenous graft will be recognized and attacked by immune cells (such as NK cells) in the host, and then inhibit or eliminate the donor, resulting in host-versus-graft reaction (HVGR). For example, in allogeneic cell transplantation, when the HLA-I molecules of allogeneic cells are deleted, the host CD8+-mediated cellular immune rejection can be reduced. In one example, the present invention provides immune cells with low or no expression of endogenous HLA-II/B2M.
移植物抗宿主病(GVHD)是由于外源移植供体T淋巴细胞的TCR的多样性，以及与宿 主HLA分子的不兼容性，供体T淋巴细胞会识别宿主正常组织上的抗原，经扩增并释放一系列细胞因子，大大增强了移植物对宿主抗原的免疫反应，攻击宿主细胞。在一实例中，本发明提供内源性HLA-II/TCR低表达或不表达的免疫细胞。在一实例中，本发明采用CRISPR系统敲除内源性TCR的α链的基因TRAC制备得到内源性TCR低表达或不表达的细胞。Graft-versus-host disease (GVHD) is due to the diversity of TCR in T lymphocytes of exogenously transplanted donors, and the interaction with host Due to the incompatibility of the main HLA molecules, the donor T lymphocytes will recognize the antigens on the normal tissues of the host, amplify and release a series of cytokines, which greatly enhance the immune response of the graft to the host antigens and attack the host cells. In one example, the present invention provides immune cells with low or no expression of endogenous HLA-II/TCR. In one example, the present invention uses the CRISPR system to knock out the gene TRAC of the alpha chain of the endogenous TCR to prepare cells with low or no expression of the endogenous TCR.
在靶细胞(例如表达靶抗原的肿瘤细胞)反复刺激下，外源移植物的供体免疫细胞中内源性NKG2A表达上调，会被本发明组合物中识别NKG2A的免疫细胞杀伤。此外，NKG2A低表达或不表达可能解除免疫细胞本身的抑制作用，从而发挥更强的抗肿瘤能力。在一实例中，本发明提供内源性HLA-II/NKG2A低表达或不表达的免疫细胞。Under repeated stimulation of target cells (such as tumor cells expressing target antigens), the expression of endogenous NKG2A in donor immune cells of exogenous grafts is up-regulated, and will be killed by immune cells that recognize NKG2A in the composition of the present invention. In addition, low expression or no expression of NKG2A may release the inhibitory effect of immune cells themselves, thus exerting stronger anti-tumor ability. In one example, the present invention provides immune cells with low or no expression of endogenous HLA-II/NKG2A.
上述免疫细胞不显著活化异体免疫细胞。上述免疫细胞能降低异体免疫排斥反应。The above-mentioned immune cells did not significantly activate allogeneic immune cells. The above-mentioned immune cells can reduce the allogeneic immune rejection.
上述识别肿瘤抗原的免疫细胞和/或识别NKG2A多肽和肿瘤抗原的免疫细胞，能显著杀伤肿瘤细胞，且不显著活化异体免疫细胞。上述识别肿瘤抗原的免疫细胞和/或识别NKG2A多肽和肿瘤抗原的免疫细胞，能显著杀伤肿瘤细胞，且异体免疫排斥反应低。The above-mentioned immune cells that recognize tumor antigens and/or immune cells that recognize NKG2A polypeptides and tumor antigens can significantly kill tumor cells without significantly activating allogeneic immune cells. The above-mentioned immune cells that recognize tumor antigens and/or immune cells that recognize NKG2A polypeptides and tumor antigens can significantly kill tumor cells with low allogeneic immune rejection.
药物组合物pharmaceutical composition
本发明的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，还可包含药学上可接受的载体。The antibodies, immune conjugates containing the antibodies, chimeric receptors, and host cells of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents. In addition to the effective amount of the antibody, immune conjugate, chimeric receptor, nucleic acid or host cell, the composition may also contain a pharmaceutically acceptable carrier.
术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
在一些实施方案中，所述组合物包含另一治疗剂。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些和/或其药学上可接受的盐或类似物。在一些实施方案中，所述治疗剂包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述另外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。在一些实施方案中，所述另外的治疗剂包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂等。In some embodiments, the composition includes another therapeutic agent. In some embodiments, the other therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof. In some embodiments, such therapeutic agents include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and norvibin(TM) (vinorelbine, 5'-dehydrogensulfide); topoisomerase I inhibitors, such as camptothecin compounds, including Camptosar™ (irinotecan HCL), Hycamtin™ (topotecan HCL), and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, Examples include etoposide, teniposide, and midoxezoz; alkylating agents cisplatin, cyclophosphamide, mechlorethamine, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, briginazine, uracil mustard, cloprofen, and dacarbazine; antimetabolites, including cytarabine, 5-fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine; antibiotics, including but not limited to doxorubicin, bleomycin , dactinomycin, daunorubicin, mymycin, mitomycin, sarcomycin C, and daunorubicin; and other chemotherapy drugs, including but not limited to antitumor antibodies, dacarbazine, azacytidine, amsacam, melphalan, ifosfamide, and mitoxantrone. In some embodiments, the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil. In some embodiments, such additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides), and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2, among others.
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和 蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。Specific examples of some substances that can be used as pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and Sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.
本文所述的药物组合物可包含一种或多种药学可接受的盐。“药学可接受的盐”指这样一种盐，其保留亲本化合物的期望生物学活性且不产生任何不利的毒物学效果(参见例如，Berge,S.M等人.,1977,J.Pharm.Sci.66:1-19)。此类盐的例子包括酸加成盐和碱加成盐。The pharmaceutical compositions described herein may comprise one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, eg, Berge, S.M et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
酸加成盐包括衍生自无毒无机酸，诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等的盐，以及衍生自无毒有机酸，诸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳香族酸、脂肪族和芳香族磺酸等的盐。碱加成盐包括衍生自碱土金属(诸如钠、钾、镁、钙等)的盐，以及衍生自无毒有机胺的盐，诸如N,N'-二苄乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等。Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, and those derived from nontoxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
本文所述的药物组合物还可包含抗氧化剂。抗氧化剂的实例包括但不限于:水溶性抗氧化剂，诸如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，诸如抗坏血酸棕榈酸酯、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)，卵磷脂、没食子酸丙酯、α-生育酚等；和金属螯合剂，诸如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。The pharmaceutical compositions described herein may also include antioxidants. Examples of antioxidants include, but are not limited to: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, alcohol Petroleum acid, phosphoric acid, etc.
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。The composition of the present invention can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dosage beneficial to the patient. The mode of administration can be, for example, parenteral administration (such as injection) or other therapeutic modes.
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。"Parenteral" administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本发明组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。In some embodiments, compositions may be isotonic, ie, they may have the same osmotic pressure as blood and tear fluid. The desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. The viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickener, if desired. Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of thickener will depend on the agent chosen. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, eg a liquid dosage form.
试剂盒Reagent test kit
本发明还提供了包含本文所述的抗体、免疫缀合物、嵌合受体、核酸或宿主细胞的试剂盒。在一些实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的本文所述的 抗体、嵌合受体、核酸或宿主细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，所述试剂盒包含本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，以及将本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞给予个体的说明书。说明书中通常包含使用本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞来治疗或预防癌症或肿瘤的方法。在一些实施方案中，试剂盒包含本文所述的宿主细胞，并且可以包括约1×104个细胞至约1×106个细胞。在一些实施方案中，试剂盒可以包括至少约1×105个细胞，至少约1×106个细胞，至少约1×107个细胞，至少约4×107个细胞，至少约5×107个细胞，至少约6×107个细胞，至少约6×107个细胞，8×107个细胞，至少约9×107个细胞，至少约1×108个细胞，至少约2×108个细胞，至少约3×108个细胞，至少约4×108个细胞，至少约5×108个细胞，至少约6×108个细胞，至少约6×108细胞，至少约8×108个细胞，至少约9×108细胞，至少约1×109个细胞，至少约2×109个细胞，至少约3×109个细胞，至少约4×109个细胞，至少约5×109个细胞，至少约6×109个细胞，至少约8×109个细胞，至少约9×109个细胞，至少约1×1010个细胞，至少约2×1010个细胞，至少约3×1010个细胞，至少约4×1010个细胞，至少约5×1010个细胞，至少约6×1010个细胞，至少约7×1010个细胞、至少约8×1010个细胞、至少约9×1010个细胞，至少约1×1011个细胞，至少约2×1011个细胞，至少约3×1011个细胞，至少约4×1011个细胞，至少约5×1011个细胞，至少约8×1011个细胞，至少约9×1011个细胞，或至少约1×1012个细胞。例如，可以在试剂盒中包括大约5×1010个细胞。在另一个实例中，试剂盒可以包括3×106个细胞；细胞可以扩增至约5×1010个细胞并施用于受试者。The invention also provides kits comprising the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein. In some embodiments, a kit may comprise an effective amount of a compound described herein comprising one or more unit dosage forms. Therapeutic or prophylactic compositions of antibodies, chimeric receptors, nucleic acids or host cells. In some embodiments, kits comprise sterile containers that may contain therapeutic or prophylactic compositions; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, foil, or other materials suitable for holding medications. In some embodiments, the kit comprises an antibody, immune conjugate, chimeric receptor, nucleic acid or host cell described herein, and instructions for administering the antibody, immune conjugate, chimeric receptor, nucleic acid or host cell described herein to an individual. The instructions generally include methods of treating or preventing cancer or tumors using the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein. In some embodiments, the kit comprises a host cell described herein, and can include from about 1 x 104 cells to about 1 x 106 cells.在一些实施方案中，试剂盒可以包括至少约1×10 5个细胞，至少约1×10 6个细胞，至少约1×10 7个细胞，至少约4×10 7个细胞，至少约5×10 7个细胞，至少约6×10 7个细胞，至少约6×10 7个细胞，8×10 7个细胞，至少约9×10 7个细胞，至少约1×10 8个细胞，至少约2×10 8个细胞，至少约3×10 8个细胞，至少约4×10 8个细胞，至少约5×10 8个细胞，至少约6×10 8个细胞，至少约6×10 8细胞，至少约8×10 8个细胞，至少约9×10 8细胞，至少约1×10 9个细胞，至少约2×10 9个细胞，至少约3×10 9个细胞，至少约4×10 9个细胞，至少约5×10 9个细胞，至少约6×10 9个细胞，至少约8×10 9个细胞，至少约9×10 9个细胞，至少约1×10 10个细胞，至少约2×10 10个细胞，至少约3×10 10个细胞，至少约4×10 10个细胞，至少约5×10 10个细胞，至少约6×10 10个细胞，至少约7×10 10个细胞、至少约8×10 10个细胞、至少约9×10 10个细胞，至少约1×10 11个细胞，至少约2×10 11个细胞，至少约3×10 11个细胞，至少约4×10 11个细胞，至少约5×10 11个细胞，至少约8×10 11个细胞，至少约9×10 11个细胞，或至少约1×10 12个细胞。 For example, approximately 5 x 1010 cells can be included in the kit. In another example, the kit can include 3 x 106 cells; the cells can be expanded to about 5 x 1010 cells and administered to the subject.
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。In some embodiments, the kit can include allogeneic cells. In some embodiments, a kit can include a cell that can contain a genomic modification. In some embodiments, a kit may comprise "ready-to-use" cells. In some embodiments, a kit can include cells that can be expanded for clinical use. In some cases, kits may contain contents intended for research purposes.
在一些实施方案中，说明书包括以下中的至少一个：治疗剂的描述；用于治疗或预防肿瘤或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本发明所述的抗体用于治疗或预防肿瘤的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给与本发明的抗体的方法。In some embodiments, the instructions include at least one of: a description of the therapeutic agent; dosage regimen and administration for treating or preventing a tumor or symptoms thereof; precautions, warnings, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and/or citations. Instructions may be printed directly on the container (if present), or as a label on the container, or as a separate sheet, booklet, card or folder provided within or in the container. In some embodiments, the instructions provide a method of administering an antibody described herein for treating or preventing a tumor. In certain instances, the instructions provide for administering an antibody of the invention before, after, or concurrently with administration of a chemotherapeutic agent.
用于诊断/检测/治疗的方法Methods for Diagnosis/Detection/Treatment
术语“调控”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。在一具体实施方式中，是指负向改变。 The term "modulate" refers to positive or negative changes. Examples of adjustments include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes. In a specific embodiment, refers to a negative change.
术语“治疗”是指在试图改变疾病过程的干预措施，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。The term "treatment" refers to interventions in an attempt to modify the disease process, either prophylaxis or intervention in the clinicopathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing down the progression of the disease, improving or relieving the disease, remission or improving the prognosis, etc.
术语“预防”是指在试图在疾病(如细胞移植产生的排斥反应)产生前进行的干预措施。The term "prevention" refers to interventions that are attempted in advance of a disease such as rejection of a cell transplant.
术语“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指肿瘤。The term "tumor antigen" refers to an antigen emerging or overexpressed during the onset, progression of a hyperproliferative disease. In certain aspects, a hyperproliferative disorder of the invention refers to a tumor.
本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。The tumor antigens described in the present invention may be solid tumor antigens or blood tumor antigens.
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)； ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。优选的，所述肿瘤抗原为CS1、Claudin18.2、GPC3、BCMA或者CD19。The tumor antigens of the present invention include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; B7H6; KIT (CD117); Interleukin 13 receptor subunit alpha (IL-13Rα); Interleukin 11 receptor alpha (IL-11Rα); Prostate stem cell antigen (PSCA); Prostate-specific membrane antigen (PSMA); Carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Tyrosinase; Mesothelin; ); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); IX (CAIX); LMP2; Ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); 7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; 1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; Polysialic acid; placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternative reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); Oncoma testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); Immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); Bone marrow stromal cell antigen 2 (BST2); ; Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is CS1, Claudin18.2, GPC3, BCMA or CD19.
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。Pathogen antigens are selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; virus antigens are selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.
本文中使用的术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。The term "individual" as used herein refers to any animal, such as a mammal or a marsupial. Subjects of the invention include, but are not limited to, humans, non-human primates (such as rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
本文所用的术语“有效量”是指提供治疗或预防益处的量。As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.
本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒都可以用于治疗方法中。Any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein can be used in methods of treatment.
在一个方面，提供用作药物的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒。在另一方面，提供用于治疗疾病的任一抗NKG2A抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，提供用于治疗方法的任一抗NKG2A抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，本发明提供任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的宿主细胞、药物组合物或试剂盒，其用于治疗患有疾病的个体的方法，所述方法包括向个体施用有效量的任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的免疫宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。In one aspect, any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit for use as a medicament is provided. In another aspect, any anti-NKG2A antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in treating a disease is provided. In certain embodiments, any anti-NKG2A antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition, or kit for use in a method of treatment is provided. In certain embodiments, the invention provides any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified host cell, pharmaceutical composition or kit for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual an effective amount of any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor modified immune host cell, pharmaceutical composition or kit. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" is preferably a human.
在另一个方面，本发明提供任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒在制备或配制药物中的用途。在一个实施方案中，所述药物用于治疗疾病。在另一个实施方案中，所述药物用于治疗疾病的方法，所述方法包括向患病个体施用有效量的药物。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。 In another aspect, the present invention provides the use of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit in the preparation or formulation of a medicament. In one embodiment, the medicament is used to treat a disease. In another embodiment, the medicament is for use in a method of treating a disease comprising administering an effective amount of the medicament to a diseased individual. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" is preferably a human.
在另一个方面，本发明提供用于治疗疾病的方法。在一个实施方案中，所述方法包括向患有表达HLA-E的疾病的个体施用有效量的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。In another aspect, the invention provides methods for treating a disease. In one embodiment, the method comprises administering to an individual having an HLA-E expressing disease an effective amount of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" is preferably a human.
在另一个方面，本发明提供例如用于任一上述治疗方法的包含本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒的药物制剂。在一个实施方案中，所述药物制剂包含本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒和药用载体。在另一个实施方案中，所述药物制剂包含本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒和至少一种另外的治疗剂。In another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein, eg, for use in any of the aforementioned methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits and pharmaceutically acceptable carriers provided herein. In another embodiment, the pharmaceutical formulation comprises any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits provided herein and at least one additional therapeutic agent.
在另一个方面，所述药物制剂用于治疗疾病。在一个实施方案中，向患病个体施用所述药物制剂。根据任一以上实施方案的“个体”优选是人。In another aspect, the pharmaceutical formulation is used to treat a disease. In one embodiment, the pharmaceutical formulation is administered to a diseased individual. An "individual" according to any of the above embodiments is preferably a human.
在另一个方面，本发明提供用于制备药物或药物制剂的方法，所述方法包括将本文中提供的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒与药用载体混合，例如，以用于任一上述治疗方法。在一个实施方案中，用于制备药物或药物制剂的方法还包括添加至少一种另外的治疗剂至药物或药物制剂。In another aspect, the present invention provides a method for the preparation of a medicament or a pharmaceutical preparation, the method comprising mixing any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit provided herein with a pharmaceutically acceptable carrier, e.g., for use in any of the aforementioned methods of treatment. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.
本发明的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒可以单独地用于治疗或与其他试剂组合地用于治疗。例如，本发明的任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的宿主细胞、药物组合物或试剂盒可以与至少一种另外的治疗剂共同施用。Any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits of the invention can be used in therapy alone or in combination with other agents. For example, any of the anti-NKG2A antibodies, immunoconjugates, chimeric antigen receptor modified host cells, pharmaceutical compositions or kits of the invention can be co-administered with at least one additional therapeutic agent.
上述的此种组合治疗包括组合施用(其中两种以上治疗剂被包含在同一或分开的制剂中)和分开施用，在此种情况中，本发明的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒的施用可以发生在另外的治疗剂或试剂的施用之前、同时、和/或之后。在一个实施方案中，本发明的任一抗NKG2A抗体、免疫缀合物、嵌合抗原受体修饰的宿主细胞、药物组合物或试剂盒的施用和另外的治疗剂的施用彼此发生在约一个月以内、或在约一周、两周或三周以内，或在约一天、两天、三天、四天、五天或六天以内。Such combination therapy as described above includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case the administration of any anti-NKG2A antibody, immunoconjugate, host cell, pharmaceutical composition or kit of the invention may occur before, simultaneously with, and/or after the administration of the additional therapeutic agent or agent. In one embodiment, administration of any anti-NKG2A antibody, immunoconjugate, chimeric antigen receptor-modified host cell, pharmaceutical composition or kit of the invention and administration of the additional therapeutic agent occur within about one month, or within about one, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days of each other.
本发明的任一抗NKG2A抗体、免疫缀合物、宿主细胞、药物组合物或试剂盒(以及任意另外的治疗剂)可以通过任何合适的手段施用，包括肠胃外施用、肺内施用或鼻内施用，以及，如果具备治疗需要，病变内施用。肠胃外输注包括肌肉内施用、静脉内施用、动脉内施用、腹膜内施用或皮下施用。用药可以是通过任何合适的途径，例如，通过注射，如静脉内或皮下注射，这部分取决于施用是短暂的还是长期的。本文中考虑多种用药方案，包括但不限于单次施用或在多个时间点的多次施用、推注施用，和脉冲注入。Any of the anti-NKG2A antibodies, immunoconjugates, host cells, pharmaceutical compositions or kits of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary or intranasal administration, and, if therapeutically warranted, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. A variety of dosing regimens are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常， 施用包含约1×104至约1×1010个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×105至约1×109个免疫反应性细胞、约5×105至约5×108个免疫反应性细胞、或约1×106至约1×107个免疫反应性细胞。然而，根据肿瘤的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。A formulation comprising a population of immunoreactive cells administered to an individual comprises a plurality of immunoreactive cells effective to treat and/or prevent a particular indication or disease. Thus, a therapeutically effective population of immunoreactive cells can be administered to an individual. usually, A formulation comprising about 1 x 104 to about 1 x 1010 immunoreactive cells is administered. In most cases, the formulation will contain about 1 x 105 to about 1 x 109 immunoreactive cells, about 5 x 105 to about 5 x 108 immunoreactive cells, or about 1 x 106 to about 1 x 107 immunoreactive cells. However, depending on the location, origin, identity, extent and severity of the tumor, the age and physical condition of the individual to be treated, etc., the number of CAR immunoreactive cells administered to an individual will vary between wide ranges. Your doctor will ultimately determine the proper dosage to use.
在一些实施方案中，使用嵌合受体来刺激宿主细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一些实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。In some embodiments, chimeric receptors are used to stimulate host cell-mediated immune responses. For example, a T cell-mediated immune response is an immune response that involves the activation of T cells. Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells displaying foreign antigen epitopes on their surface, such as cancer cells displaying tumor antigens. In other embodiments, chimeric antigen receptors are used to provide anti-tumor immunity in mammals. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.
在某些情况下，治疗患有肿瘤的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的宿主细胞。所述宿主细胞可结合肿瘤靶分子并诱导癌细胞死亡。如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的宿主细胞。In certain instances, methods of treating a subject with a tumor may involve administering to the subject in need of treatment one or more host cells described herein. The host cells can bind tumor target molecules and induce cancer cell death. As previously stated, the present invention also provides a method of treating a pathogenic infection in an individual comprising administering to said individual a therapeutically effective amount of a host cell of the invention.
本发明的宿主细胞的给药频率将根据包括所治疗疾病的因素、特定宿主细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的宿主细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的宿主细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。The frequency of administration of the host cells of the invention will depend on factors including the disease being treated, elements of the particular host cell and the mode of administration. For example, it can be administered 4 times a day, 3 times, 2 times a day, or once a day, every other day, every three days, every four days, every five days, once every six days, once a week, once every eight days, once every nine days, every ten days, once a week, or twice a month. As described herein, due to the improved viability of the host cells of the present application, they can not only be administered in a lower therapeutically effective amount than similar host cells that do not express exogenous type I interferon, but also can be administered at a lower frequency to obtain at least similar and preferably more significant curative effects.
本发明的优点：Advantages of the present invention:
1.本发明提供了特异性结合NKG2A的抗体，是全人源抗体，免疫原性低，可能的临床副反应少；1. The present invention provides an antibody specifically binding to NKG2A, which is a fully human antibody with low immunogenicity and few possible clinical side effects;
2.本发明的抗体能够有效阻断肿瘤细胞的HLA-E与NK细胞的NKG2A/CD94结合，降低了表达HLA-E的肿瘤细胞通过NKG2A/CD94通路对NK细胞的抑制作用，增强NK细胞对肿瘤细胞的杀伤作用，表现出良好的抗肿瘤效果。2. The antibody of the present invention can effectively block the combination of HLA-E of tumor cells and NKG2A/CD94 of NK cells, reduce the inhibitory effect of tumor cells expressing HLA-E on NK cells through the NKG2A/CD94 pathway, enhance the killing effect of NK cells on tumor cells, and show good anti-tumor effects.
3.表达本发明的抗体制备的NKG2A-CAR的T细胞能杀伤NK细胞；利用本发明的抗体制备的既靶向肿瘤又靶向NK细胞的串联CAR的T细胞能杀伤NK细胞，也能增强其抗肿瘤作用；表达本发明的抗体制备的NKG2A-CAR的UCAR-T细胞能抵抗NK细胞的杀伤，增强其存活能力，并且能协同靶向肿瘤的T细胞或CAR-T细胞的抗肿瘤作用。3. T cells expressing NKG2A-CAR prepared by the antibody of the present invention can kill NK cells; T cells prepared by using the antibody of the present invention targeting both tumors and NK cells can kill NK cells and enhance their anti-tumor effect; UCAR-T cells expressing NKG2A-CAR prepared by the antibody of the present invention can resist the killing of NK cells, enhance their survival ability, and can cooperate with the anti-tumor effect of tumor-targeting T cells or CAR-T cells.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。 Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, generally follow the conventional conditions such as the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or the conditions suggested by the manufacturer.
实施例1.NKG2A抗体的筛选和鉴定Example 1. Screening and identification of NKG2A antibodies
1、使用全人噬菌体展示文库筛选针对NKG2A特异的抗体1. Screening for NKG2A-specific antibodies using a fully human phage display library
(1)NKG2A/CD94、NKG2C/CD94异源二聚体的制备(1) Preparation of NKG2A/CD94, NKG2C/CD94 heterodimers
利用真核表达质粒V152S构建分别表达NKG2A/CD94、NKG2C/CD94异源二聚体的载体。将包括mFc(序列如SEQ ID NO：84所示)、NKG2A胞外段(序列如SEQ ID NO：71所示)、(G4S)3(序列如SEQ ID NO：110所示)、CD94胞外段(序列如SEQ ID NO：77所示)的片段mFc-NKG2A-CD94插入真核表达质粒V152S，构建成载体V152S-mFc-NKG2A-CD94；将包括mFc(序列如SEQ ID NO：84所示)、NKG2C胞外段(序列如73所示)、(G4S)3(序列如SEQ ID NO：110所示)、CD94胞外段(序列如SEQ ID NO：77所示)的片段mFc-NKG2C-CD94插入真核表达质粒V152S，构建成载体V152S-mFc-NKG2C-CD94(见图1)。The eukaryotic expression plasmid V152S was used to construct vectors expressing NKG2A/CD94 and NKG2C/CD94 heterodimers respectively.将包括mFc(序列如SEQ ID NO：84所示)、NKG2A胞外段(序列如SEQ ID NO：71所示)、(G 4 S) 3 (序列如SEQ ID NO：110所示)、CD94胞外段(序列如SEQ ID NO：77所示)的片段mFc-NKG2A-CD94插入真核表达质粒V152S，构建成载体V152S-mFc-NKG2A-CD94；将包括mFc(序列如SEQ ID NO：84所示)、NKG2C胞外段(序列如73所示)、(G 4 S) 3 (序列如SEQ ID NO：110所示)、CD94胞外段(序列如SEQ ID NO：77所示)的片段mFc-NKG2C-CD94插入真核表达质粒V152S，构建成载体V152S-mFc-NKG2C-CD94(见图1)。
将载体V152S-mFc-NKG2A-CD94、V152S-mFc-NKG2C-CD94分别转染HEK293细胞(中国典型培养物保藏中心(CCTCC))后培养7天，培养液离心取上清，使用Mabselect Sure柱亲和纯化分别得到抗原NKG2A/CD94、NKG2C/CD94异源二聚体。The vectors V152S-mFc-NKG2A-CD94 and V152S-mFc-NKG2C-CD94 were respectively transfected into HEK293 cells (China Center for Type Culture Collection (CCTCC)) and cultured for 7 days. The culture medium was centrifuged to obtain the supernatant, and the Mabselect Sure column was used for affinity purification to obtain antigen NKG2A/CD94 and NKG2C/CD94 heterodimers, respectively.
(2)NKG2A抗体的筛选(2) Screening of NKG2A antibody
本发明使用的噬菌体展示文库为本公司构建的噬菌体文库，库容为1E+11。利用本领域技术人员已知的筛选方法得到针对NKG2A/CD94异源二聚体高度特异的Fab片段。The phage display library used in the present invention is a phage library constructed by our company with a capacity of 1E+11. A highly specific Fab fragment for NKG2A/CD94 heterodimer was obtained using screening methods known to those skilled in the art.
简言之，分别包被上述制备的异源二聚体10μg/ml抗原mFc-NKG2A-CD94、mFc-NKG2C-CD94于免疫管。用含有2％脱脂奶粉的磷酸缓冲液(MPBS)室温静置封闭2小时。为了筛选到特异性结合NKG2A的抗体，将噬菌体文库加入包被mFc-NKG2C-CD94的免疫管中结合1小时。取上清液加入包被mFc-NKG2A-CD94的免疫管中结合1.5小时，随后将非特异性的噬菌体洗掉，将结合的噬菌体洗脱下来并感染对数生长期的大肠杆菌TG1。扩大培养洗脱下来的噬菌体，并使用PEG/NaCl沉淀纯化扩大后的噬菌体文库用于下一轮的筛选。筛选一共进行三轮，富集与NKG2A/CD94异源二聚体特异性结合的Fab噬菌体克隆。通过针对NKG2A的标准ELISA方法确定阳性克隆。ELISA使用mFc-NKG2C-CD94作为无关抗原来验证抗体的特异性。一共筛选了1504个克隆，其中50个克隆只结合mFc-NKG2A-CD94，不结合mFc-NKG2C-CD94。经过测序，得到了2个克隆。表达纯化这2个克隆，得到Fab形式的抗体A1和A2。In short, 10 μg/ml antigens mFc-NKG2A-CD94 and mFc-NKG2C-CD94 prepared above were coated on immunotubes respectively. Block with phosphate-buffered saline (MPBS) containing 2% skimmed milk powder at room temperature for 2 hours. In order to screen for antibodies specifically binding to NKG2A, the phage library was added to an immunotube coated with mFc-NKG2C-CD94 for 1 hour. The supernatant was taken and added to an immunotube coated with mFc-NKG2A-CD94 for 1.5 hours, then the non-specific phages were washed away, the bound phages were eluted and infected with Escherichia coli TG1 in logarithmic growth phase. The eluted phages were expanded and purified by PEG/NaCl precipitation for the next round of screening. A total of three rounds of screening were performed to enrich the Fab phage clones specifically binding to the NKG2A/CD94 heterodimer. Positive clones were identified by standard ELISA methods against NKG2A. Antibody specificity was verified by ELISA using mFc-NKG2C-CD94 as an irrelevant antigen. A total of 1504 clones were screened, among which 50 clones only bound to mFc-NKG2A-CD94 and did not bind to mFc-NKG2C-CD94. After sequencing, 2 clones were obtained. These two clones were expressed and purified to obtain antibodies A1 and A2 in Fab form.
A1的HCDR1的氨基酸序列如SEQ ID NO:1所示、HCDR2的氨基酸序列如SEQ ID NO:2所示、HCDR3的氨基酸序列如SEQ ID NO:3所示、LCDR1的氨基酸序列如SEQ ID NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:6所示。A1的重链可变区的氨基酸序列如SEQ ID NO:18所示、轻链可变区的氨基酸序列如SEQ ID NO:20所示。A1的重链的氨基酸序列如SEQ ID NO：34所示，轻链的氨基酸序列如SEQ ID NO：42所示。A2的HCDR1的氨基酸序列如SEQ ID NO:7所示、HCDR2的氨基酸序列如SEQ ID NO:8所示、HCDR3的氨基酸序列如SEQ ID NO:9所示、LCDR1的氨基酸序列如SEQ ID  NO:4所示、LCDR2的氨基酸序列如SEQ ID NO:5所示、LCDR3的氨基酸序列如SEQ ID NO:10所示。A2的重链可变区的氨基酸序列如SEQ ID NO:22所示、轻链可变区的氨基酸序列如SEQ ID NO:24所示。A2重链的氨基酸序列如SEQ ID NO：35所示，轻链的氨基酸序列如SEQ ID NO：46所示。The amino acid sequence of HCDR1 of A1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6. The amino acid sequence of the heavy chain variable region of A1 is shown in SEQ ID NO:18, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:20. The amino acid sequence of the heavy chain of A1 is shown in SEQ ID NO:34, and the amino acid sequence of the light chain is shown in SEQ ID NO:42. The amino acid sequence of HCDR1 of A2 is shown in SEQ ID NO: 7, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 9, and the amino acid sequence of LCDR1 is shown in SEQ ID As shown in NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:10. The amino acid sequence of the heavy chain variable region of A2 is shown in SEQ ID NO:22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:24. The amino acid sequence of the heavy chain of A2 is shown in SEQ ID NO:35, and the amino acid sequence of the light chain is shown in SEQ ID NO:46.
同时构建和真核表达纯化出IgG4形式的抗体，得到两个特异性结合NKG2A/CD94异源二聚体的抗体，命名为A1-IgG4和A2-IgG4。A1-IgG4的重链氨基酸序列如SEQ ID NO:40所示，轻链氨基酸序列如SEQ ID NO:42所示。A2-IgG4的重链氨基酸序列如SEQ ID NO:44所示，轻链氨基酸序列如SEQ ID NO:46所示。Simultaneous construction and eukaryotic expression of purified IgG4 antibodies resulted in two antibodies specifically binding to NKG2A/CD94 heterodimers, named A1-IgG4 and A2-IgG4. The heavy chain amino acid sequence of A1-IgG4 is shown in SEQ ID NO:40, and the light chain amino acid sequence is shown in SEQ ID NO:42. The heavy chain amino acid sequence of A2-IgG4 is shown in SEQ ID NO:44, and the light chain amino acid sequence is shown in SEQ ID NO:46.
2、通过标准ELISA检测抗体A1和A2的特异性2. Detect the specificity of antibodies A1 and A2 by standard ELISA
抗原mFc-NKG2A-CD94和mFc-NKG2C-CD94分别用PBS稀释至浓度为5μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后分别加入一抗A1(10μg/ml)或A2(10μg/ml)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Flag-HRP，1:4000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。NA为空白对照。结果见图2，抗体A1、A2均特异性结合NKG2A/CD94异源二聚体，不结合NKG2C/CD94异源二聚体。Antigens mFc-NKG2A-CD94 and mFc-NKG2C-CD94 were diluted with PBS to a concentration of 5 μg/ml, and coated overnight at 4°C. 2% MPBS (skimmed milk powder/PBS) was blocked at room temperature for 1 hour; after washing with PBS for 3 times, primary antibody A1 (10 μg/ml) or A2 (10 μg/ml) was added, and incubated for 1 hour at room temperature; after washing with PBST for 5 times, secondary antibody (anti-Flag-HRP, 1:4000, Sigma) was added to continue incubation at room temperature for 1 hour; after washing with PBST for 5 times, TMB was developed and the OD450 value was read with a microplate reader. NA is the blank control. The results are shown in Figure 2. Antibodies A1 and A2 both specifically bind to NKG2A/CD94 heterodimer, but not to NKG2C/CD94 heterodimer.
3、利用ELISA分别测定抗体A1-IgG4、A2-IgG4与NKG2A/CD94异源二聚体的EC503. Use ELISA to measure the EC50 of antibodies A1-IgG4, A2-IgG4 and NKG2A/CD94 heterodimer
上述制备的mFc-NKG2A-CD94用PBS稀释至浓度为2.5μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后分别加入一抗(A1-IgG4、A2-IgG4:10μg/ml开始5倍稀释8个梯度)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Fc-HRP，1:10000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。随后通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。结果见图3，抗体A1-IgG4和A2-IgG4均与NKG2A/CD94异源二聚体结合，且呈现浓度梯度依赖性，EC50值分别为0.012μg/ml，0.011μg/ml。The mFc-NKG2A-CD94 prepared above was diluted with PBS to a concentration of 2.5 μg/ml, and coated overnight at 4°C. Block with 2% MPBS (skimmed milk powder/PBS) at room temperature for 1 hour; wash with PBS for 3 times, add primary antibody (A1-IgG4, A2-IgG4: 5-fold dilution and 8 gradients starting from 10 μg/ml), and incubate at room temperature for 1 hour; wash with PBST for 5 times, add secondary antibody (anti-Fc-HRP, 1:10000, Sigma) and continue to incubate at room temperature for 1 hour; wash with PBST for 5 times, develop color with TMB and use a microplate reader Read the OD450 value. Then, GraphPad Prism5 software was used to perform four-parameter fitting with the primary antibody concentration as the abscissa and the OD450 value as the ordinate to calculate the EC50 value. The results are shown in Figure 3. Antibodies A1-IgG4 and A2-IgG4 both bind to NKG2A/CD94 heterodimers in a concentration gradient-dependent manner, with EC50 values of 0.012 μg/ml and 0.011 μg/ml, respectively.
4、利用Biacore测定抗体A1-IgG4和A2-IgG4的亲和力4. Use Biacore to determine the affinity of antibodies A1-IgG4 and A2-IgG4
用Anti-huFc antibody包被芯片CM5(ID:180925-0245:1738291)，将A1-IgG4和A2-IgG4抗体分别作为配体，mFc-NKG2A-CD94作为流动相(图4中的曲线从上到下分别代表浓度为150nM、50nM、16.67nM、5.56nM和1.85nM)，再生试剂为3M MgCl2，25℃，实验数据用Biacore T200Evaluation Software2.0软件处理。选择“surface bound”，Rmax选择“local”，使用1:1的langmuir模型进行拟合。结果见图4，A1-IgG4和A2-IgG4结合NKG2A/CD94异源二聚体的亲和力KD值分别为4.41nM和4.30nM。Chip CM5 (ID: 180925-0245: 1738291) was coated with Anti-huFc antibody, A1-IgG4 and A2-IgG4 antibodies were used as ligands, and mFc-NKG2A-CD94 was used as mobile phase (the curves in Figure 4 represent concentrations of 150nM, 50nM, 16.67nM, 5.56nM and 1. 85nM), the regeneration reagent was 3M MgCl 2 , at 25°C, and the experimental data were processed with Biacore T200Evaluation Software2.0 software. Select "surface bound", select "local" for Rmax, and use a 1:1 langmuir model for fitting. The results are shown in Figure 4, the affinity KD values of A1-IgG4 and A2-IgG4 binding to NKG2A/CD94 heterodimer were 4.41nM and 4.30nM, respectively.
5、利用FACs测定抗体A1-IgG4、A2-IgG4与靶细胞结合的特异性5. Using FACs to determine the specificity of binding of antibodies A1-IgG4 and A2-IgG4 to target cells
利用常规分子生物学技术，通过pWPT慢病毒载体介导的方式将CD94全长(包含CD94全长，Flag标签)、NKG2A-CD94(包含NKG2A全长，F2A，CD94全长，Flag标签)、NKG2C-CD94(包含NKG2C全长，F2A，CD94全长，Flag标签)、NKG2E-CD94(包含NKG2E全长，F2A，CD94全长，Flag标签)，分别转入CHO-K1细胞(也称为CHOK1)构建成过表达目的基因的混合克隆细 胞株，使用抗Flag抗体确认混合克隆株构建成功后用有限稀释法进行单克隆筛选，最后成功得到表达CD94的CHO-K1稳转细胞株CHOK1-CD94、表达NKG2A-CD94的CHO-K1稳转细胞株CHOK1-NKG2A-CD94、表达NKG2C-CD94的CHO-K1稳转细胞株CHOK1-NKG2C-CD94及表达NKG2E-CD94的CHO-K1稳转细胞株CHOK1-NKG2E-CD94(载体图见图5)，阳性率均大于90％。Using conventional molecular biology techniques, CD94 full-length (including CD94 full-length, Flag tag), NKG2A-CD94 (including NKG2A full-length, F2A, CD94 full-length, Flag tag), NKG2C-CD94 (including NKG2C full-length, F2A, CD94 full-length, Flag tag), NKG2E-CD94 (including NKG2 E full length, F2A, CD94 full length, Flag tag), respectively transferred into CHO-K1 cells (also known as CHOK1) to construct a mixed clone cell overexpressing the target gene Cell lines, use anti-Flag antibody to confirm the successful construction of mixed clones, and then perform monoclonal screening by limiting dilution method. Finally, the CHO-K1 stable cell line CHOK1-CD94 expressing CD94, the CHO-K1 stable cell line CHOK1-NKG2A-CD94 expressing NKG2A-CD94, the CHO-K1 stable cell line CHOK1-NKG2C-CD94 expressing NKG2C-CD94 and NKG2C-CD94 were successfully obtained. The G2E-CD94 CHO-K1 stably transfected cell line CHOK1-NKG2E-CD94 (see Figure 5 for the vector map), and the positive rates were all greater than 90%.
其中CD94全长碱基序列为SEQ ID No.128，NKG2A全长碱基序列为SEQ ID No.69，NKG2C全长碱基序列为SEQ ID No.124，NKG2E全长碱基序列为SEQ ID No.126，Flag标签碱基序列为SEQ ID No.112，F2A碱基序列为SEQ ID No.108。The full-length base sequence of CD94 is SEQ ID No.128, the full-length base sequence of NKG2A is SEQ ID No.69, the full-length base sequence of NKG2C is SEQ ID No.124, the full-length base sequence of NKG2E is SEQ ID No.126, the base sequence of the Flag tag is SEQ ID No.112, and the base sequence of F2A is SEQ ID No.108 .
分别取CHOK1-NKG2A-CD94，CHOK1-NKG2C-CD94，CHOK1-NKG2E-CD94和CHOK1-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1-IgG4、A2-IgG4:5μg/mL)和二抗(Anti-Fc-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。结果见图6，抗体A1-IgG4、A2-IgG4分别结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞，不结合过表达NKG2C/CD94、NKG2E/CD94以及CD94的CHO-K1细胞。Count CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94, CHOK1-NKG2E-CD94, and CHOK1-CD94 cells and spread them on a U-shaped bottom plate, with about 2× 105 cells per well. ImmunoResearch) incubation, followed by flow cytometer for fluorescence intensity detection. The results are shown in Figure 6. Antibodies A1-IgG4 and A2-IgG4 respectively bind to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer, but do not bind to CHO-K1 cells overexpressing NKG2C/CD94, NKG2E/CD94 and CD94.
实施例2.抗体的亲和力成熟Example 2. Affinity maturation of antibodies
1、利用噬菌体展示技术进行亲和力成熟1. Affinity maturation using phage display technology
分别以A1和A2为亲本抗体，采用常规生物学技术分别构建两个噬菌体文库，一个随机化轻链的CDR1以及CDR2，另一个随机化重链的CDR1以及CDR2。然后针对抗原进行筛选，通过ELISA技术等筛选出高亲和力的抗体，即A1或A2的变体。Using A1 and A2 as parental antibodies, two phage libraries were constructed using conventional biological techniques, one randomized the CDR1 and CDR2 of the light chain, and the other randomized the CDR1 and CDR2 of the heavy chain. Then screen against the antigen, and screen out high-affinity antibodies, that is, variants of A1 or A2, by ELISA technology and the like.
首先基于抗体A1(Fab)构建模板质粒。对于轻链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和IL1R(核酸序列如SEQ ID NO：91所示)，PCR扩增片段1；使用引物IL2F(核酸序列如SEQ ID NO：92所示)和FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段2；然后通过搭桥PCR连接片段1和片段2得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为2.6ⅹ109。对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和F10H1R(核酸序列如SEQ ID NO：87所示)，PCR扩增片段3；使用引物F10H2F(核酸序列如SEQ ID NO：88所示)和FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段4；然后通过搭桥PCR连接片段3和片段4得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为3.2ⅹ109。First, a template plasmid was constructed based on antibody A1 (Fab). For light chain CDR1 and CDR2 randomized phage library, use primer LMF (nucleic acid sequence as shown in SEQ ID NO: 85) and IL1R (nucleic acid sequence as shown in SEQ ID NO: 91), PCR amplifies fragment 1; Use primer IL2F (nucleic acid sequence as shown in SEQ ID NO: 92) and FdR (nucleic acid sequence as shown in SEQ ID NO: 86), PCR amplifies fragment 2; The full-length Fab sequence was digested with NcoI and NotI, ligated into the same digested template plasmid by T4 ligase, and electrotransformed into TG1 competent cells with a storage capacity of 2.6ⅹ10 9 .对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和F10H1R(核酸序列如SEQ ID NO：87所示)，PCR扩增片段3；使用引物F10H2F(核酸序列如SEQ ID NO：88所示)和FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段4；然后通过搭桥PCR连接片段3和片段4得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为3.2ⅹ10 9 。
抗体A2亲和力成熟文库的构建与A1类似，基于抗体A2(Fab)构建模板质粒。使用与A1相同的引物随机化轻链的CDR1和CDR2，得到的噬菌体文库库容为1.89ⅹ109。对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF(核酸序列如SEQ ID NO：85所示)和BH1R(核酸序列如SEQ ID NO：89所示)，PCR扩增片段5；使用引物BH2F(核酸序列如SEQ ID NO：90所示)和 FdR(核酸序列如SEQ ID NO：86所示)，PCR扩增片段6；然后通过搭桥PCR连接片段5和片段6得到含有随机化序列的Fab全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为1.22ⅹ109。The construction of the antibody A2 affinity maturation library is similar to that of A1, and the template plasmid is constructed based on the antibody A2 (Fab). Using the same primers as A1 to randomize CDR1 and CDR2 of the light chain, the resulting phage library has a capacity of 1.89ⅹ10 9 . For the heavy chain CDR1 and CDR2 randomized phage library, use primers LMF (nucleic acid sequence as shown in SEQ ID NO: 85) and BH1R (nucleic acid sequence as shown in SEQ ID NO: 89), PCR amplifies fragment 5; use primers BH2F (nucleic acid sequence as shown in SEQ ID NO: 90) and FdR (the nucleic acid sequence is shown in SEQ ID NO: 86), fragment 6 was amplified by PCR; then fragment 5 and fragment 6 were connected by bridging PCR to obtain the full-length Fab containing the randomized sequence, and then the full-length fragment was digested with NcoI and NotI, ligated into the same digested template plasmid by T4 ligase, and electrotransformed into TG1 competent cells with a storage capacity of 1.22ⅹ10 9 .
2、噬菌体文库的筛选2. Screening of phage library
参照实施例1方法，进行两轮筛选。第一轮筛选用浓度为5μg/ml的抗原mFc-NKG2A-CD94包被，并使用浓度为5μg/ml的抗原mFc-NKG2C-CD94清除；第二轮筛选用浓度为1μg/ml的抗原mFc-NKG2A-CD94包被，同时使用浓度为2μg/ml的抗原mFc-NKG2E-CD94清除。两轮筛选后通过ELISA确定阳性克隆，共挑取79个阳性克隆进行测序，并选取22个克隆原核表达纯化得到Fab抗体。最终得到4个克隆A3、A4、A5、A6。经测序，抗体的序列如表4、5所示.Referring to the method in Example 1, two rounds of screening were carried out. The first round of screening was coated with the antigen mFc-NKG2A-CD94 at a concentration of 5 μg/ml and cleared with the antigen mFc-NKG2C-CD94 at a concentration of 5 μg/ml; the second round of screening was coated with the antigen mFc-NKG2A-CD94 at a concentration of 1 μg/ml and cleared with the antigen mFc-NKG2E-CD94 at a concentration of 2 μg/ml. After two rounds of screening, positive clones were determined by ELISA. A total of 79 positive clones were selected for sequencing, and 22 clones were selected for prokaryotic expression and purification to obtain Fab antibodies. Finally, 4 clones A3, A4, A5 and A6 were obtained. After sequencing, the sequences of the antibodies are shown in Tables 4 and 5.
表4抗体VH和VL序列
Table 4 Antibody VH and VL sequences
表5抗体CDR序列
Table 5 Antibody CDR sequence
表6抗体重链和轻链序列
Table 6 Antibody heavy chain and light chain sequences
将这4个克隆构建成IgG4形式，进行表达纯化。经测序，4个抗体的序列如表7所示。 These 4 clones were constructed into IgG4 format, and expression and purification were carried out. After sequencing, the sequences of the four antibodies are shown in Table 7.
表7抗体(huIgG4)序列
Table 7 antibody (huIgG4) sequence
如图7所示，比较了6个抗体A1、A2、A3、A4、A5和A6的重链可变区的氨基酸序列。经测序，A3与母本A1的重链可变区序列相似性为98.3％。A4与母本A2的重链可变区序列相似性为94.8％。A5与母本A2的重链可变区序列相似性为94.8％。A6与母本A2的重链可变区序列相似性为93.1％。As shown in Fig. 7, the amino acid sequences of the heavy chain variable regions of six antibodies A1, A2, A3, A4, A5 and A6 were compared. After sequencing, the heavy chain variable region sequence similarity between A3 and parent A1 was 98.3%. The heavy chain variable region sequence similarity between A4 and the parent A2 was 94.8%. The heavy chain variable region sequence similarity between A5 and the parent A2 was 94.8%. The heavy chain variable region sequence similarity between A6 and the parent A2 was 93.1%.
3、利用FACs测定抗体(Fab形式)与靶细胞结合的特异性3. Using FACs to determine the specificity of antibody (Fab format) binding to target cells
分别取CHOK1-NKG2A-CD94，CHOK1-NKG2C-CD94和CHOK1-NKG2E-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1、A2、A3、A4、A5、A6:10μg/mL)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。结果见图8，6个抗体(Fab形式)都特异性结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞，不结合过表达NKG2C/CD94以及NKG2E/CD94的CHO-K1细胞。Count CHOK1-NKG2A-CD94, CHOK1-NKG2C-CD94 and CHOK1-NKG2E-CD94 cells and spread them on a U-shaped bottom plate, with about 2× 105 cells per well, and then conduct primary antibody (A1, A2, A3, A4, A5, A6: 10 μg/mL) and secondary antibody (Anti-Fab-FITC: 1:200, Jackson ImmunoRe search) and then detected by flow cytometry. The results are shown in Figure 8. All six antibodies (in Fab format) specifically bind to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer, but not to CHO-K1 cells overexpressing NKG2C/CD94 and NKG2E/CD94.
4、利用ELISA测定抗体(Fab形式)与NKG2A/CD94异源二聚体的EC504. Determination of EC50 between antibody (Fab format) and NKG2A/CD94 heterodimer by ELISA
抗原mFc-NKG2A-CD94用PBS稀释至浓度为2μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后分别加入一抗(A3、A4、A5、A6:25μg/mL起始5倍梯度稀释8个梯度)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Flag-HRP，1：4000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。随后通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。结果见图9，4个抗体(Fab形式)均与NKG2A/CD94异源二聚体结合，且呈现浓度梯度依赖性，亲和力成熟后的抗体的结合能力明显优于对应的母本抗体。EC50值如表8所示。The antigen mFc-NKG2A-CD94 was diluted with PBS to a concentration of 2 μg/ml, and coated overnight at 4°C. 2% MPBS (skimmed milk powder/PBS) was blocked at room temperature for 1 hour; after washing with PBS for 3 times, primary antibodies were added (A3, A4, A5, A6: 25 μg/mL initial 5-fold gradient dilution and 8 gradients), and incubated at room temperature for 1 hour; after washing with PBST for 5 times, secondary antibody (anti-Flag-HRP, 1:4000, Sigma) was added to continue incubation at room temperature for 1 hour; after washing with PBST for 5 times, TMB was developed and OD was read with a microplate reader 450 values. Then, GraphPad Prism5 software was used to perform four-parameter fitting with the primary antibody concentration as the abscissa and the OD450 value as the ordinate to calculate the EC50 value. The results are shown in Figure 9. All four antibodies (Fab format) bind to the NKG2A/CD94 heterodimer in a concentration gradient-dependent manner, and the binding ability of the antibody after affinity maturation is significantly better than that of the corresponding parental antibody. The EC50 values are shown in Table 8.
表8抗体(Fab形式)与NKG2A/CD94异源二聚体结合的EC50值
Table 8 The EC50 value of antibody (Fab form) binding to NKG2A/CD94 heterodimer
5、利用FACs测定抗体(Fab形式)与靶细胞结合的EC505. Using FACs to determine the EC50 of antibody (Fab format) binding to target cells
取CHOK1-NKG2A-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1、A2、A3、A4、A5、A6:25μg/mL起始5倍梯度稀释8个梯度)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。实验数据用FlowJo分析软件计算平均荧光强度(MFI)再根据“校准MFI＝实测MFI-阴性对照MFI”计算各浓度点校准MFI，随后通过GraphPad Prism5软件以一抗浓度为横坐标，校准平均荧光强度(MFI)为纵坐标进行四参数拟合，计算EC50值。结果见图10，抗体(Fab形式)结合过表达NKG2A/CD94异源 二聚体的CHO-K1细胞的EC50值如表9所示，亲和力成熟后的抗体胞的结合能力明显优于对应的母本抗体。CHOK1-NKG2A-CD94 cells were counted and spread on a U-shaped bottom plate, with about 2× 105 cells per well, and then incubated with primary antibody (A1, A2, A3, A4, A5, A6: 25 μg/mL initial 5-fold gradient dilution and 8 gradients) and secondary antibody (Anti-Fab-FITC: 1:200, Jackson ImmunoResearch), and then detected the fluorescence intensity with a flow cytometer. The experimental data was calculated with FlowJo analysis software and the mean fluorescence intensity (MFI) was calculated, and then the calibration MFI of each concentration point was calculated according to "calibration MFI=measured MFI-negative control MFI". Then, the four-parameter fitting was performed with the primary antibody concentration as the abscissa and the calibrated mean fluorescence intensity (MFI) as the ordinate through the GraphPad Prism5 software, and the EC50 value was calculated. The results are shown in Figure 10, the antibody (Fab format) binds to the overexpressed NKG2A/CD94 heterologous The EC50 values of the dimerized CHO-K1 cells are shown in Table 9, and the binding ability of the antibody cells after affinity maturation is significantly better than that of the corresponding parent antibody.
表9抗体(Fab形式)与过表达NKG2A/CD94异源二聚体的CHO-K1细胞结合的EC50
Table 9 EC50 of antibody (Fab format) binding to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer
实施例3.抗体(IgG4形式)与抗原的结合活性检测Example 3. Detection of binding activity of antibody (IgG4 format) to antigen
1、利用ELISA检测抗体(IgG4形式)与NKG2A/CD94异源二聚体的结合活性1. Use ELISA to detect the binding activity of antibody (IgG4 format) to NKG2A/CD94 heterodimer
抗原mFc-NKG2A-CD94用PBS稀释至浓度为2μg/ml，4℃包被过夜。2％MPBS(脱脂奶粉/PBS)室温封闭1h；PBS清洗3遍后加入一抗(A1-IgG4、A2-IgG4、A3-IgG4、A4-IgG4、A5-IgG4、A6-IgG4:5μg/mL起始5倍梯度稀释8个梯度)，室温孵育1h；PBST清洗5遍后加入二抗(anti-Fc-HRP，1：10000，Sigma)继续室温孵育1h；PBST清洗5遍后TMB显色并用酶标仪读数OD450值。通过GraphPad Prism5软件以一抗浓度为横坐标，OD450值为纵坐标进行四参数拟合，计算EC50值。结果见图11，抗体(IgG4形式)与NKG2A/CD94异源二聚体结合，且呈现浓度梯度依赖性，EC50值如表10所示。The antigen mFc-NKG2A-CD94 was diluted with PBS to a concentration of 2 μg/ml, and coated overnight at 4°C. Block with 2% MPBS (skimmed milk powder/PBS) at room temperature for 1 h; wash with PBS for 3 times, add primary antibody (A1-IgG4, A2-IgG4, A3-IgG4, A4-IgG4, A5-IgG4, A6-IgG4: 5 μg/mL initial 5-fold serial dilution of 8 gradients), incubate at room temperature for 1 h; wash with PBST for 5 times, add secondary antibody (anti-Fc-HRP, 1:10 000, Sigma) and continue to incubate at room temperature for 1 h; after washing with PBST for 5 times, the TMB color develops and the OD450 value is read with a microplate reader. Using the GraphPad Prism5 software, the primary antibody concentration was used as the abscissa, and the OD450 value was used as the ordinate to perform four-parameter fitting to calculate the EC50 value. The results are shown in Figure 11. The antibody (IgG4 format) binds to the NKG2A/CD94 heterodimer in a concentration gradient-dependent manner, and the EC50 values are shown in Table 10.
表10抗体(IgG4形式)与NKG2A/CD94异源二聚体结合的EC50值
The EC50 value of table 10 antibody (IgG4 form) combined with NKG2A/CD94 heterodimer
2、利用FACS检测抗体(IgG4形式)与靶细胞的结合2. Use FACS to detect the binding of antibody (IgG4 format) to target cells
取CHOK1-NKG2A-CD94细胞计数铺U型底板，每孔约2×105个细胞，然后分别进行一抗(A1-IgG4、A2-IgG4、A3-IgG4、A4-IgG4、A5-IgG4、A6-IgG4:25μg/mL起始5倍梯度稀释8个梯度)和二抗(Anti-Fab-FITC:1:200,Jackson ImmunoResearch)孵育，随后用流式分析仪进行荧光强度检测。实验数据用FlowJo分析软件计算平均荧光强度(MFI)再根据“校准MFI＝实测MFI-阴性对照MFI”计算各浓度点校准MFI，随后通过GraphPad Prism5软件以一抗浓度为横坐标，校准平均荧光强度(MFI)为纵坐标进行四参数拟合，计算EC50值。结果见图12，抗体(IgG4形式)均结合过表达NKG2A/CD94异源二聚体的CHO-K1细胞，且呈现浓度梯度依赖性，EC50值如表11所示，这表明亲和力成熟后的抗体的结合活性均有提高。The CHOK1-NKG2A-CD94 cells were counted and laid on a U-shaped bottom plate, with about 2× 105 cells per well, and then the primary antibody (A1-IgG4, A2-IgG4, A3-IgG4, A4-IgG4, A5-IgG4, A6-IgG4: 25 μg/mL initial 5-fold serial dilution 8 gradients) and secondary antibody (Anti-Fab-FITC: 1:200 , Jackson ImmunoResearch) incubation, followed by flow cytometer for fluorescence intensity detection. The experimental data was calculated with FlowJo analysis software and the mean fluorescence intensity (MFI) was calculated, and then the calibration MFI of each concentration point was calculated according to "calibration MFI=measured MFI-negative control MFI". Then, the four-parameter fitting was performed with the primary antibody concentration as the abscissa and the calibrated mean fluorescence intensity (MFI) as the ordinate through the GraphPad Prism5 software, and the EC50 value was calculated. The results are shown in Figure 12. The antibodies (IgG4 format) all bind to CHO-K1 cells overexpressing NKG2A/CD94 heterodimers in a concentration gradient-dependent manner. The EC50 values are shown in Table 11, which indicates that the binding activity of the antibodies after affinity maturation is improved.
表11抗体(IgG4形式)与过表达NKG2A/CD94异源二聚体的CHO-K1细胞结合的EC50
Table 11 The EC50 of antibody (IgG4 form) binding to CHO-K1 cells overexpressing NKG2A/CD94 heterodimer
3、利用Biacore检测抗体(IgG4形式)与NKG2A/CD94异源二聚体的亲和力3. Use Biacore to detect the affinity of antibody (IgG4 format) to NKG2A/CD94 heterodimer
用Anti-huFc antibody包被芯片CM5(ID:180925-0245:1738291)，将A3-IgG4、A4-IgG4、A5-IgG41、A6-IgG4抗体作为配体，mFc-NKG2A-CD94作为流动相(图13中的曲线从上到下分别代表浓度为100nM、33.33nM、11.11nM、3.70nM和1.23nM)，再生试剂为3M MgCl2，25℃。实验数据用Biacore T200 Evaluation Software2.0软件处理。选择“surface bound”，Rmax选择 “local”，使用1:1的langmuir模型进行拟合。结果见图13，所得抗体(IgG4形式)与NKG2A/CD94异源二聚体的结合亲和力KD值如表12所示，经过亲和力成熟后的抗体的亲和力与母本抗体相比有约12～18倍的提高。Chip CM5 (ID: 180925-0245: 1738291) was coated with Anti-huFc antibody, A3-IgG4, A4-IgG4, A5-IgG41, A6-IgG4 antibodies were used as ligands, mFc-NKG2A-CD94 was used as mobile phase (the curves in Figure 13 from top to bottom represent concentrations of 100 nM, 33.33 nM, 11.11 nM, 3.70 nM and 1.23 nM), the regeneration reagent is 3M MgCl 2 , 25°C. The experimental data were processed with Biacore T200 Evaluation Software2.0 software. Select "surface bound", select Rmax "local", using a 1:1 Langmuir model for fitting. The results are shown in Figure 13. The KD value of the binding affinity between the obtained antibody (IgG4 format) and the NKG2A/CD94 heterodimer is shown in Table 12, and the affinity of the antibody after affinity maturation is about 12-18 times higher than that of the parental antibody.
表12抗体(IgG4形式)与NKG2A/CD94异源二聚体结合的亲和力
Table 12 Antibody (IgG4 form) binds to the affinity of NKG2A/CD94 heterodimer
实施例4.NKG2A的天然配体HLA-E四聚体的制备及活性检测Example 4. Preparation and Activity Detection of the Natural Ligand HLA-E Tetramer of NKG2A
1、NKG2A的天然配体HLA-E四聚体的制备1. Preparation of natural ligand HLA-E tetramer of NKG2A
利用常规分子生物学技术，通过质粒转导的方式将分别包含片段HLA-E-avi-(包含人HLA-E胞外段(序列如SEQ ID NO:79所示)、avi标签(序列如SEQ ID NO:114所示)、片段β2m(序列如SEQ ID NO:81所示)的质粒pET22b-HLA-E、pET22b-β2m，分别转入BL21菌株(载体见图14)。Using conventional molecular biology techniques, the plasmids pET22b-HLA-E and pET22b-β2m comprising the fragment HLA-E-avi- (comprising the extracellular segment of human HLA-E (sequence shown in SEQ ID NO: 79), the avi tag (sequence shown in SEQ ID NO: 114), and fragment β2m (sequence shown in SEQ ID NO: 81) were respectively transferred into BL21 strains (see Figure 1 for the vector) by plasmid transduction. 4).
使用BL21菌株原核诱导表达，收集菌体再超声破碎后收集包涵体沉淀。包涵体沉淀经过洗涤后再用8M尿素溶解，经过阴离子交换柱的进一步纯化后电泳检测，纯度90％以上。100ml复性液中加入5mg VMA九肽(氨基酸序列如SEQ ID NO:82所示，吉尔生化合成)，再以1:2的摩尔比加入纯化的HLA-E和β2m，得到复性复合物，再用PBS 5％甘油透析。透析后产物经过分子筛纯化，收集目的峰后浓缩并置换到10mMTris pH8.0，分装-80℃保存。使用birA酶对复合物进行生物素标记，标记产物和SA-PE(PE标记的SA抗体(BD HorizonTM))1:4的比例轻柔混合，得到最终的HLA-E四聚体。Prokaryotic induction of BL21 strain was used to induce expression, and the bacteria were collected and then ultrasonically disrupted to collect inclusion body precipitates. The inclusion body precipitate was washed and then dissolved with 8M urea, further purified by an anion exchange column and detected by electrophoresis, with a purity of over 90%. Add 5 mg of VMA nonapeptide (amino acid sequence as shown in SEQ ID NO: 82, synthesized by Jill Biochemical) to 100 ml of refolding solution, and then add purified HLA-E and β2m at a molar ratio of 1:2 to obtain a refolding complex, which is then dialyzed against PBS 5% glycerol. After dialysis, the product was purified by molecular sieves, the target peak was collected, concentrated and replaced to 10mM Tris pH 8.0, and stored at -80°C. The complex was biotin-labeled with birA enzyme, and the labeled product and SA-PE (PE-labeled SA antibody (BD Horizon TM )) were gently mixed at a ratio of 1:4 to obtain the final HLA-E tetramer.
2、将纯化后的HLA-E四聚体与内源性表达NKG2A/CD94的原代NK细胞进行结合活性检测2. Detection of binding activity between purified HLA-E tetramer and primary NK cells endogenously expressing NKG2A/CD94
NK细胞通过NK Cell Isolation Kit(Miltenyi Biotec)从外周血PBMCs中纯化得到，并用含500IU/ml IL-2和150IU/ml IL-15的NKMedium(Miltenyi Biotec)培养8天后收集得到NKG2A阳性NK细胞(NKG2A+NK)，阳性率为86％。HLA-E四聚体使用浓度从5μg/ml开始，5倍梯度稀释，空白孔浓度为0，与NK细胞4℃共孵育45min后，用含1％FBS的PBS洗3遍，随后用流式细胞仪检测PE荧光信号。结果见图15，HLA-E四聚体与表达NKG2A/CD94的NK细胞有明显结合，且呈现浓度梯度依赖性，EC50为0.1029μg/ml。NK cells were purified from peripheral blood PBMCs by NK Cell Isolation Kit (Miltenyi Biotec), and treated with NK cells containing 500IU/ml IL-2 and 150IU/ml IL-15 Medium (Miltenyi Biotec) was cultured for 8 days to collect NKG2A positive NK cells (NKG2A + NK), and the positive rate was 86%. The concentration of HLA-E tetramer started from 5 μg/ml, 5-fold serial dilution, and the concentration of the blank hole was 0. After co-incubating with NK cells at 4°C for 45 min, they were washed 3 times with PBS containing 1% FBS, and then the PE fluorescence signal was detected by flow cytometry. The results are shown in Figure 15. The HLA-E tetramer was significantly combined with the NK cells expressing NKG2A/CD94 in a concentration-gradient-dependent manner, with an EC50 of 0.1029 μg/ml.
实施例5.检测NKG2A抗体(IgG4形式)阻断NKG2A与其配体HLA-E的结合Example 5. Detection of NKG2A antibody (IgG4 format) blocking the binding of NKG2A to its ligand HLA-E
取实施例4中制备的NKG2A阳性的NK细胞(NKG2A+NK)，同时加入HLA-E-PE和抗NKG2A抗体在4℃共孵育45min，检测PE荧光信号强度。根据实施例4，HLA-E-PE浓度选用EC50值，即0.103μg/ml，抗体浓度从10μg/ml开始，5倍梯度稀释至0.000128μg/ml，空白组只含HLA-E-PE。共孵育结束后，用含1％FBS的PBS洗3遍，随后通过流式细胞分析仪进行PE荧光信号检测，得到MFI值，抑制率计算公式为：(MFI(空白组)-MFI(实验组))/MFI(空白组)×100％。结果见图16，所得亲和力成熟抗体在1μg/ml以上浓度时均可完全抑制HLA-E 四聚体与NK细胞的结合，其中半数抑制有效浓度IC50值如表13所示。实验表明亲和力成熟抗体竞争抑制HLA-E四聚体与NK细胞结合的能力相比较母本有明显提高。Take the NKG2A-positive NK cells (NKG2A + NK) prepared in Example 4, add HLA-E-PE and anti-NKG2A antibody at the same time and incubate at 4° C. for 45 minutes to detect the intensity of PE fluorescence signal. According to Example 4, the EC50 value of HLA-E-PE concentration was selected, that is, 0.103 μg/ml, the antibody concentration started from 10 μg/ml, and was diluted to 0.000128 μg/ml in a 5-fold gradient, and the blank group only contained HLA-E-PE. After co-incubation, wash 3 times with PBS containing 1% FBS, and then detect PE fluorescence signal by flow cytometer to obtain MFI value. The results are shown in Figure 16. The obtained affinity matured antibody can completely inhibit HLA-E at a concentration above 1 μg/ml The IC50 value of the half inhibitory effective concentration for the combination of tetramer and NK cells is shown in Table 13. Experiments show that the ability of affinity maturation antibody to inhibit the combination of HLA-E tetramer and NK cells is significantly improved compared with that of the mother.
表13抗体(IgG4形式)阻断HLA-E与NK细胞结合的IC50值
Table 13 Antibodies (IgG4 form) block the IC50 value of HLA-E binding to NK cells
实施例6.NKG2A抗体能够降低表达HLA-E的靶细胞对NK细胞活性的抑制Example 6. NKG2A antibody can reduce the inhibition of NK cell activity by target cells expressing HLA-E
取实施例4中制备的NKG2A+NK细胞为效应细胞。靶细胞选用不表达HLA-E的K562细胞(人髓性白血病细胞，中科院细胞库)、过表达HLA-E的K562细胞(称为K562-HLA-E细胞)以及内源性表达HLA-E的FaDu细胞(人咽鳞状细胞，ATCC)，HLA-E表达水平见图17。通过慢病毒介导的方式将人HLA-E胞外区(序列如SEQ ID NO:78所示)转入K562细胞得到K562-HLA-E细胞。The NKG2A + NK cells prepared in Example 4 were taken as effector cells. The target cells are K562 cells that do not express HLA-E (human myeloid leukemia cells, Chinese Academy of Sciences Cell Bank), K562 cells that overexpress HLA-E (called K562-HLA-E cells), and FaDu cells that endogenously express HLA-E (human pharyngeal squamous cells, ATCC). The expression levels of HLA-E are shown in Figure 17. The extracellular region of human HLA-E (sequence shown in SEQ ID NO: 78) was transferred into K562 cells by lentivirus-mediated method to obtain K562-HLA-E cells.
将1×105个NK细胞与三种靶细胞分别按照效靶比1:1共孵育，同时加入CD107a-APC抗体(购自BD Biosciences，5μl/test)及NKG2A抗体(IgG4形式，10μg/ml，空白组不添加NKG2A抗体)共孵育1h，添加蛋白转运抑制剂(Brefeldin A/Monensin Mix)继续孵育3h。收集细胞，在实验组添加带PE-Cy7荧光的CD56抗体(CD56-PE-Cy7，购自eBioscience，5μl/test，用于检测NK细胞)和带FITC荧光的抗Fc抗体(anti-Fc-FITC抗体，购自Jackson Immunoresearch，1:200，用于检测NKG2A阳性NK细胞)共孵育后进行FACs检测，空白组添加CD56-PE-Cy7和anti-NKG2A-PE抗体(购自Miltenyi Biotec,2μl/test)共孵育后进行FACs检测，分析各组NKG2A+NK细胞中CD107a的表达情况。结果见图18，NKG2A+NK细胞与K562细胞共孵育后，CD107a表达水平较高，说明肿瘤细胞K562没有结合NK细胞上的NKG2A而抑制NK细胞活性，因此添加NKG2A抗体对CD107a表达水平无显著影响。而NKG2A+NK细胞与K562-HLA-E细胞、或与FaDu细胞共孵育后，CD107a表达水平较低，说明肿瘤细胞上的HLA-E与NK细胞上的NKG2A结合能够明显抑制NK细胞活性；而添加NKG2A抗体竞争性地与NK细胞上的NKG2A结合，阻断NK细胞上的NKG2A与肿瘤细胞上的HLA-E结合，从而降低HLA-E高表达肿瘤细胞对NK细胞活性抑制，因此相对于没有添加抗体组，添加NKG2A抗体各组的CD107a表达水平明显提高。由于FaDu细胞内源性表达HLA-E的量较低，导致CD107a表达水平提高程度低于HLA-E高表达组K562-HLA-E。1×10 5 NK cells were co-incubated with the three target cells according to the effect-to-target ratio of 1:1. At the same time, CD107a-APC antibody (purchased from BD Biosciences, 5 μl/test) and NKG2A antibody (IgG4 form, 10 μg/ml, blank group without NKG2A antibody) were added for 1 hour, and protein transport inhibitor (Brefeldin A/Monensin Mix) was added for 3 hours. Cells were collected, and in the experimental group, CD56 antibody with PE-Cy7 fluorescence (CD56-PE-Cy7, purchased from eBioscience, 5 μl/test, used to detect NK cells) and anti-Fc antibody with FITC fluorescence (anti-Fc-FITC antibody, purchased from Jackson Immunoresearch, 1:200, used to detect NKG2A-positive NK cells) were added to the experimental group and incubated for FACs detection. The blank group was added with CD56-PE-Cy7 and ant After co-incubation with i-NKG2A-PE antibody (purchased from Miltenyi Biotec, 2 μl/test), FACs detection was performed to analyze the expression of CD107a in NKG2A + NK cells in each group. The results are shown in Figure 18. After co-incubation of NKG2A + NK cells and K562 cells, the expression level of CD107a was higher, indicating that the tumor cell K562 did not bind to NKG2A on NK cells to inhibit the activity of NK cells. Therefore, the addition of NKG2A antibody had no significant effect on the expression level of CD107a. After NKG2A + NK cells were co-incubated with K562-HLA-E cells or FaDu cells, the expression level of CD107a was low, indicating that the combination of HLA-E on tumor cells and NKG2A on NK cells can significantly inhibit the activity of NK cells; adding NKG2A antibody competitively binds to NKG2A on NK cells, blocking the combination of NKG2A on NK cells and HLA-E on tumor cells, thereby reducing HLA- E high-expression tumor cells can inhibit the activity of NK cells, so compared with the group without antibody addition, the expression level of CD107a in each group added with NKG2A antibody was significantly increased. Due to the low endogenous expression of HLA-E in FaDu cells, the increase in CD107a expression level was lower than that in the HLA-E high expression group K562-HLA-E.
实施例7.NKG2A抗体增强原代NK细胞对表达HLA-E的靶细胞的杀伤作用Example 7. NKG2A antibody enhances the killing effect of primary NK cells on target cells expressing HLA-E
同实施例6，将6×104个NKG2A+NK细胞分别与靶细胞(K562，K562-HLA-E和FaDu细胞)共孵育，效靶比为3:1，同时加入10μg/ml NKG2A抗体(IgG4形式,空白组不添加抗体)，共孵育4h后收集培养基上清，通过LDH检测法计算杀伤作用。As in Example 6, 6×10 4 NKG2A + NK cells were co-incubated with target cells (K562, K562-HLA-E and FaDu cells) respectively, with an effect-to-target ratio of 3:1. At the same time, 10 μg/ml NKG2A antibody (IgG4 format, no antibody was added to the blank group) was added. After co-incubating for 4 hours, the supernatant of the medium was collected, and the killing effect was calculated by LDH detection method.
按照下面公式计算杀伤率：Calculate the kill rate according to the following formula:
细胞毒性％＝[实验组LDH释放量(Avg.)–效应细胞自发LDH释放量(Avg.)–靶 细胞自发LDH释放量(Avg.)]/[靶细胞最大LDH释放量(Avg.)-靶细胞自发LDH释放量(Avg.)–体积校准(Avg.)]×100％Cytotoxicity% = [LDH release amount of experimental group (Avg.) - spontaneous LDH release amount of effector cells (Avg.) - target Spontaneous LDH release of cells (Avg.)]/[maximum LDH release of target cells (Avg.)-spontaneous LDH release of target cells (Avg.)-volume calibration (Avg.)]×100%
结果见图19，NKG2A+NK细胞对K562细胞有约55％杀伤率，对高表达HLA-E的K562-HLA-E细胞和FaDu细胞的杀伤率均低于20％。说明了靶细胞上表达的HLA-E与NK细胞上表达的NKG2A结合，抑制NK细胞活性，从而降低NK细胞对靶细胞的杀伤。添加NKG2A抗体后可以阻断靶细胞的HLA-E与NK细胞的NKG2A结合，降低靶细胞对NK细胞活性抑制，从而提高NK细胞活性，提高NK细胞杀伤高表达HLA-E的肿瘤细胞。The results are shown in Figure 19. NKG2A + NK cells have a killing rate of about 55% on K562 cells, and the killing rates on K562-HLA-E cells and FaDu cells highly expressing HLA-E are lower than 20%. It shows that the HLA-E expressed on the target cell combines with the NKG2A expressed on the NK cell to inhibit the activity of the NK cell, thereby reducing the killing of the target cell by the NK cell. The addition of NKG2A antibody can block the combination of HLA-E of target cells and NKG2A of NK cells, reduce the inhibition of NK cell activity by target cells, thereby increase the activity of NK cells, and increase the ability of NK cells to kill tumor cells with high expression of HLA-E.
实施例8.抗NKG2A特异性CAR-T细胞对NK细胞的杀伤作用Example 8. The killing effect of anti-NKG2A specific CAR-T cells on NK cells
1、抗NKG2A抗体A4、A5嵌合抗原受体质粒的构建1. Construction of anti-NKG2A antibody A4, A5 chimeric antigen receptor plasmids
以PRRLSIN-cPPT.EF-1α(购自Addgene公司)为载体，构建了表达抗体A4、A5的二代嵌合抗原受体的慢病毒质粒，即PRRLSIN-A4-BBZ和PRRLSIN-A5-BBZ。Using PRRLSIN-cPPT.EF-1α (purchased from Addgene) as a vector, lentiviral plasmids expressing second-generation chimeric antigen receptors of antibodies A4 and A5, namely PRRLSIN-A4-BBZ and PRRLSIN-A5-BBZ, were constructed.
A4-BBZ(SEQ ID NO：115)序列由CD8α信号肽、A4scFv、CD8铰链区、CD8跨膜区、CD137胞内信号传导结构域以及CD3δ顺序连接组成。A5-BBZ(SEQ ID NO：116)序列由CD8α信号肽、A5scFv、CD8铰链区、CD8跨膜区、CD137胞内信号传导结构域以及CD3δ顺序连接组成。其中，CD8α信号肽(SEQ ID NO：93)、A4scFv(SEQ ID NO：64)、A5scFv(SEQ ID NO：66)、CD8铰链区(SEQ ID NO：95)、CD8跨膜区(SEQ ID NO：97)、CD137胞内信号传导结构域(SEQ ID NO：101)、CD3δ(SEQ ID NO：105)。The A4-BBZ (SEQ ID NO: 115) sequence is composed of CD8α signal peptide, A4scFv, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signaling domain and CD3δ sequentially connected. The A5-BBZ (SEQ ID NO: 116) sequence is composed of CD8α signal peptide, A5scFv, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signaling domain and CD3δ sequentially connected. Among them, CD8α signal peptide (SEQ ID NO: 93), A4scFv (SEQ ID NO: 64), A5scFv (SEQ ID NO: 66), CD8 hinge region (SEQ ID NO: 95), CD8 transmembrane region (SEQ ID NO: 97), CD137 intracellular signaling domain (SEQ ID NO: 101), CD3δ (SEQ ID NO: 1 05).
2、NKG2A-CAR T细胞的制备2. Preparation of NKG2A-CAR T cells
用磷酸钙法进行慢病毒的包装，病毒上清用PEG8000/NaCl进行纯化，纯化后病毒按MOI值为10感染CD3/CD28磁珠活化48小时后的T细胞，分别得到表达A4-BBZ、A5-BBZ的CAR-T细胞，未转染病毒的T细胞视为UTD。感染后第6天用FACS法检测CAR阳性率，检测抗原为Bio-NKG2A-CD94，二抗为BV421标记SA抗体(BD HorizonTM)，1:200稀释使用，结果显示A4-BBZ CAR T的CAR阳性率为62.8％、A5-BBZ CAR T的CAR阳性率为59％。The calcium phosphate method was used to package the lentivirus, and the virus supernatant was purified with PEG8000/NaCl. The purified virus was infected with CD3/CD28 magnetic beads at an MOI of 10 and activated for 48 hours to obtain CAR-T cells expressing A4-BBZ and A5-BBZ. T cells that were not transfected with the virus were regarded as UTD. On the 6th day after infection, the CAR positive rate was detected by FACS method. The detection antigen was Bio-NKG2A-CD94, and the secondary antibody was BV421-labeled SA antibody (BD Horizon TM ), diluted at 1:200. The results showed that the CAR positive rate of A4-BBZ CAR T was 62.8%, and that of A5-BBZ CAR T was 59%.
3、NKG2A-CAR T细胞靶向NKG2A阳性NK细胞的体外杀伤毒性检测3. In vitro cytotoxicity detection of NKG2A-CAR T cells targeting NKG2A-positive NK cells
NK细胞通过NK Cell Isolation Kit(Miltenyi Biotec)从两位供者(#1和#2)外周血PBMCs中纯化得到，并用含500IU/ml IL-2和150IU/ml IL-15的NKMedium(Miltenyi Biotec)培养至第14天收集。取上述NK细胞分别与APC标记的NKG2A抗体(Invitrogen)(1:200稀释使用)4℃共孵育5min后，用FACS法检测NK细胞中NKG2A的表达水平。结果显示，#1供者NK细胞中NKG2A的阳性率为80.4％，#2供者NK细胞中NKG2A的阳性率为61.5％。NK cells were purified from peripheral blood PBMCs of two donors (#1 and #2) by NK Cell Isolation Kit (Miltenyi Biotec), and treated with NK cells containing 500IU/ml IL-2 and 150IU/ml IL-15 Medium (Miltenyi Biotec) was cultivated until the 14th day and collected. The above-mentioned NK cells were respectively incubated with APC-labeled NKG2A antibody (Invitrogen) (diluted at 1:200) for 5 min at 4°C, and the expression level of NKG2A in NK cells was detected by FACS method. The results showed that the positive rate of NKG2A in NK cells of #1 donor was 80.4%, and the positive rate of NKG2A in NK cells of #2 donor was 61.5%.
靶细胞：取5×104个上述NKG2A阳性的NK细胞作为靶细胞接种到96孔板。Target cells: 5×10 4 above-mentioned NKG2A positive NK cells were seeded into 96-well plates as target cells.
效应细胞：按效靶比1：1及2：1分别接种UTD细胞、A4-BBZ CAR-T细胞、A5-BBZ CAR-T细胞细胞到相应的96孔板。Effector cells: Inoculate UTD cells, A4-BBZ CAR-T cells, and A5-BBZ CAR-T cells into corresponding 96-well plates according to the effect-to-target ratio of 1:1 and 2:1, respectively.
采用流式染色法进行体外细胞杀伤实验，分别在0hr、4hr、24hr进行流式染色，检测共培养体系中NK细胞比例。结果见图20，随着共培养时间延长，UTD细胞组中NK细胞 比例无明显变化，而A4-BBZ CAR-T、A5-BBZ CAR-T组中NK细胞比例显著下降。这表明A4-BBZ CAR-T细胞和A5-BBZ CAR-T细胞均能有效杀伤表达NKG2A的NK细胞。The in vitro cell killing experiment was carried out by flow cytometry, and flow staining was performed at 0hr, 4hr, and 24hr to detect the proportion of NK cells in the co-culture system. The results are shown in Figure 20. With the prolongation of the co-culture time, the NK cells in the UTD cell group There was no significant change in the proportion, but the proportion of NK cells in the A4-BBZ CAR-T and A5-BBZ CAR-T groups decreased significantly. This indicates that both A4-BBZ CAR-T cells and A5-BBZ CAR-T cells can effectively kill NKG2A-expressing NK cells.
实施例9抗NKG2A的UCAR-T细胞能够有效抵抗NK细胞的杀伤Example 9 Anti-NKG2A UCAR-T cells can effectively resist the killing of NK cells
B2M缺失的T细胞会引起NK细胞的排斥作用，通过构建TCR/B2M缺失的NKG2A-UCAR-T细胞来验证UCAR-T细胞对NK细胞的抵抗。为避免NKG2A表达造成的自我攻击作用，同时制备了NKG2A敲除的NKG2A-UCAR-T细胞(UCAR-TKO)。B2M-deleted T cells can cause NK cell rejection, and the resistance of UCAR-T cells to NK cells was verified by constructing TCR/B2M-deleted NKG2A-UCAR-T cells. In order to avoid the self-attack effect caused by NKG2A expression, NKG2A-knockout NKG2A-UCAR-T cells (UCAR-TKO) were prepared at the same time.
体外合成靶向TCR/B2M/NKG2A基因的gRNA，序列分别如SEQ ID NO：117、118、119所示。采用常规CRISPR/Cas9技术敲除T细胞内源性TCR/B2M、或TCR/B2M/NKG2A。CRISPR/Cas 9酶(恺佧生物)和gRNA按摩尔比1：4比例进行混合，形成RNP复合物(Cas 9酶的终浓度为1uM)，室温孵育10分钟后，利用MaxCyte电转仪将RNP复合物导入到T细胞中。将实施例8中的A4-CAR-T、A5-CAR-T细胞分别进行TCR/B2M双敲除，得到A4-UCAR-T、A5-UCAR-T细胞；将A4-CAR-T、A5-CAR-T细胞分别进行TCR/B2M/NKG2A三敲除，得到A4-UCAR-T-TKO、A5-UCAR-T-TKO细胞。以同样方法敲除TCR/B2M、TCR/B2M/NKG2A基因但未转染CAR的UTD细胞(命名为UTD UCAR-T、UTD UCAR-T-TKO)进行对照，调整细胞浓度至5×105/mL，接种至96孔板，按原代扩增的#2供者NK细胞与T细胞比例1：1或1：2接种细胞，于培养箱中分别共孵育0hr、24hr、48hr。利用流式细胞术标记HLA-ABC阳性的NK细胞，检测共孵育不同时间点UCAR-T细胞比例。结果见图21，UTD UCAR-T、UTD UCAR-T-TKO细胞的比例随着时间延长而逐渐下降，表明NK细胞抑制UTD UCAR-T、UTD UCAR-T-TKO细胞生长；而表达NKG2A-CAR的UCAR-T或UCAR-T-TKO细胞在共孵育后48h比例均显著上升。这表明，抗NKG2A的UCAR-T或UCAR-T-TKO细胞能够有效抵抗NK细胞的杀伤。The gRNA targeting the TCR/B2M/NKG2A gene was synthesized in vitro, and the sequences are shown in SEQ ID NO: 117, 118, and 119, respectively. The endogenous TCR/B2M or TCR/B2M/NKG2A of T cells was knocked out by conventional CRISPR/Cas9 technology. CRISPR/Cas 9 enzyme (Kaijia Biology) and gRNA were mixed at a molar ratio of 1:4 to form an RNP complex (the final concentration of the Cas 9 enzyme was 1uM). After incubation at room temperature for 10 minutes, the RNP complex was introduced into T cells using a MaxCyte electroporator. A4-CAR-T and A5-CAR-T cells in Example 8 were respectively subjected to TCR/B2M double knockout to obtain A4-UCAR-T and A5-UCAR-T cells; A4-CAR-T and A5-CAR-T cells were respectively subjected to TCR/B2M/NKG2A triple knockout to obtain A4-UCAR-T-TKO and A5-UCAR-T-TKO cells. UTD cells (named UTD UCAR-T, UTD UCAR-T-TKO) that knocked out TCR/B2M, TCR/B2M/NKG2A genes in the same way but not transfected with CAR were used as controls, adjusted the cell concentration to 5×10 5 /mL, inoculated into 96-well plates, and inoculated the cells according to the ratio of 1:1 or 1:2 of primary amplified #2 donor NK cells to T cells, and co-incubated in the incubator for 0 hr and 24 hr, respectively. , 48hr. HLA-ABC positive NK cells were labeled by flow cytometry, and the proportion of UCAR-T cells at different time points of co-incubation was detected. The results are shown in Figure 21. The proportion of UTD UCAR-T and UTD UCAR-T-TKO cells gradually decreased with time, indicating that NK cells inhibited the growth of UTD UCAR-T and UTD UCAR-T-TKO cells; while the proportion of UCAR-T or UCAR-T-TKO cells expressing NKG2A-CAR increased significantly after 48 hours of co-incubation. This indicates that NKG2A-resistant UCAR-T or UCAR-T-TKO cells can effectively resist the killing of NK cells.
实施例10抗NKG2A的UCAR-T细胞协同靶向肿瘤抗原的CAR-T细胞的抗肿瘤作用Example 10 Anti-tumor effect of NKG2A-resistant UCAR-T cells synergistically targeting tumor antigen CAR-T cells
采用本领域常规分子生物学方法，构建靶向BCMA的嵌合抗原受体，包装慢病毒并转染T细胞，制备成靶向BCMA的CAR-T细胞。示例性，BCMA-scFv的氨基酸序列如SEQ ID NO:120所示，BCMA-CAR的氨基酸序列如SEQ ID NO:121所示。采用实施例9的方法对BCMA CAR-T细胞的B2M/TCR/NKG2A基因进行敲除得到BCMA UCAR-T细胞(命名为BCMA UCAR-T-TKO)。Using conventional molecular biology methods in the field, construct a chimeric antigen receptor targeting BCMA, package lentivirus and transfect T cells, and prepare BCMA-targeting CAR-T cells. Exemplarily, the amino acid sequence of BCMA-scFv is shown in SEQ ID NO:120, and the amino acid sequence of BCMA-CAR is shown in SEQ ID NO:121. The B2M/TCR/NKG2A gene of BCMA CAR-T cells was knocked out by the method in Example 9 to obtain BCMA UCAR-T cells (named BCMA UCAR-T-TKO).
体外培养表达BCMA的多发性骨髓瘤细胞系RPMI-8226(中国科学院细胞库)，皮下接种5×106细胞/只于NPG免疫缺陷的小鼠中(记为D0)，接种10天后瘤体积平均为200mm3左右，将小鼠分4组。D10，D14,D17,D21,D24第2、3、4组尾静脉分别注射1×106NK细胞，一共注射5次。D11，按分组尾静脉分别注射T细胞。各组具体情况如下：The BCMA-expressing multiple myeloma cell line RPMI-8226 (Cell Bank, Chinese Academy of Sciences) was cultured in vitro, and 5×10 6 cells per mouse were subcutaneously inoculated into NPG immunodeficient mice (denoted as D0). The average tumor volume was about 200 mm 3 10 days after inoculation, and the mice were divided into 4 groups. On D10, D14, D17, D21, and D24, 1×10 6 NK cells were injected into the tail vein of groups 2, 3, and 4 respectively, for a total of 5 injections. On D11, T cells were injected into the tail vein according to groups. The details of each group are as follows:
第1组：0.6×106UTD UCAR-TGroup 1: 0.6×10 6 UTD UCAR-T
第2组：0.6×106UTD UCAR-T+NKGroup 2: 0.6×10 6 UTD UCAR-T+NK
第3组：0.6×106BCMA UCAR-T-TKO+1×106A4UCAR-T+NK Group 3: 0.6×10 6 BCMA UCAR-T-TKO+1×10 6 A4UCAR-T+NK
第4组：0.6×106BCMA UCAR-T-TKO+1×106A5 UCAR-T+NKGroup 4: 0.6×10 6 BCMA UCAR-T-TKO+1×10 6 A5 UCAR-T+NK
T细胞注射后，每周2次测量体重(包括分组给药及安乐死当天)，并用游标卡尺测量并记录肿瘤长径、短径，计算肿瘤体积，根据肿瘤体积绘制肿瘤生长曲线，并比较各组间肿瘤生长曲线的差异(肿瘤体积：V＝1/2×长径×短径2)。结果见图22，在NK细胞存在下，抗NKG2A的UCAR-T细胞发挥协同BCMA UCAR-T细胞抗肿瘤作用：在D32,第3、4组小鼠体内肿瘤几乎被完全清除。After T cell injection, body weight was measured twice a week (including group administration and the day of euthanasia), and the long diameter and short diameter of the tumor were measured and recorded with a caliper, and the tumor volume was calculated. The tumor growth curve was drawn according to the tumor volume, and the difference of the tumor growth curve among the groups was compared (tumor volume: V=1/2×long diameter×short diameter 2 ). The results are shown in Figure 22. In the presence of NK cells, UCAR-T cells against NKG2A exerted synergistic anti-tumor effects with BCMA UCAR-T cells: on D32, the tumors in the mice in groups 3 and 4 were almost completely eliminated.
实施例11靶向NKG2A及肿瘤抗原的双靶点UCAR-T细胞的抗肿瘤能力及抵抗NK活性Example 11 Anti-tumor ability and anti-NK activity of dual-target UCAR-T cells targeting NKG2A and tumor antigens
以靶向肿瘤抗原BCMA为例，构建了同时靶向NKG2A和BCMA的CAR-T细胞，观察其抗肿瘤活性以及抵抗NK细胞杀伤的效果。使用载体PRRLsin，构建表达串联CAR(SEQ ID NO：122)的BCMA-NKG2A CAR-T细胞。采用实施例9的方法，对BCMA-NKG2A CAR-T细胞的B2M/TCR/NKG2A基因进行敲除得到BCMA-NKG2A UCAR-T-TKO细胞。体内外实验结果显示，靶向NKG2A及肿瘤抗原的双靶点的串联UCAR-T细胞能够有效抵抗NK细胞的杀伤，抑制肿瘤生长。Taking the tumor antigen BCMA as an example, CAR-T cells targeting both NKG2A and BCMA were constructed, and their anti-tumor activity and anti-NK cell killing effect were observed. BCMA-NKG2A CAR-T cells expressing tandem CAR (SEQ ID NO: 122) were constructed using the vector PRRLsin. Using the method in Example 9, the B2M/TCR/NKG2A gene of BCMA-NKG2A CAR-T cells was knocked out to obtain BCMA-NKG2A UCAR-T-TKO cells. The results of in vitro and in vivo experiments showed that tandem UCAR-T cells targeting NKG2A and tumor antigens could effectively resist the killing of NK cells and inhibit tumor growth.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
本申请涉及如下序列：









This application involves the following sequences:









上述序列表中当N在碱基序列中表示任意碱基。 In the above sequence listing, when N represents any base in the base sequence.

Claims (30)

  1. 识别NKG2A的全人源抗体，其特征在于，所述抗体包含轻链可变区，所述轻链可变区包含RASQSISSWLA(SEQ ID NO:4)所示的LCDR1；和/或A fully human antibody that recognizes NKG2A, characterized in that the antibody comprises a light chain variable region, and the light chain variable region comprises LCDR1 shown in RASQSISSWLA (SEQ ID NO: 4); and/or
    DASSLES(SEQ ID NO:5)所示的LCDR2；和/或LCDR2 shown in DASSLES (SEQ ID NO:5); and/or
    QQYDSYX1X2T(SEQ ID NO:129)所示的LCDR3，其中X1是I或V，X2是R或S。LCDR3 shown in QQYDSYX 1 X 2 T (SEQ ID NO: 129), wherein X 1 is I or V, and X 2 is R or S.
  2. 识别NKG2A的全人源抗体，其特征在于，所述抗体包括重链可变区，所述重链可变区选自：A fully human antibody that recognizes NKG2A, characterized in that the antibody includes a heavy chain variable region, and the heavy chain variable region is selected from:
    (1)包含SYAIS(SEQ ID NO:1)所示的HCDR1；和/或(1) comprising HCDR1 shown in SYAIS (SEQ ID NO: 1); and/or
    GIIPIFGTAX1YAQKFQG(SEQ ID NO:130)所示的HCDR2，其中X1是N或H；和/或HCDR2 shown in GIIPIFGTAX 1 YAQKFQG (SEQ ID NO: 130), wherein X 1 is N or H; and/or
    GFDGMDY(SEQ ID NO:3)所示的HCDR3；或HCDR3 represented by GFDGMDY (SEQ ID NO:3); or
    (2)包含X1X2X3X4S(SEQ ID NO:131)所示的HCDR1，其中X1是S、R或N，X2是Y、F或V，X3是A、Y或H，X4是M或V；和/或(2) HCDR1 comprising X 1 X 2 X 3 X 4 S (SEQ ID NO: 131), wherein X 1 is S, R or N, X 2 is Y, F or V, X 3 is A, Y or H, X 4 is M or V; and/or
    AIX1X2X3X4GSTYYADSVKG(SEQ ID NO:132)所示的HCDR2，其中X1是S、T或N，X2是G或A，X3是S、W、G或P，X4是G或V；和/或HCDR2 shown in AIX 1 X 2 X 3 X 4 GSTYYADSVKG (SEQ ID NO: 132), wherein X 1 is S, T or N, X 2 is G or A, X 3 is S, W, G or P, X 4 is G or V; and/or
    GYDGFDY(SEQ ID NO:9)所示的HCDR3。HCDR3 shown in GYDGFDY (SEQ ID NO:9).
  3. 如权利要求1或2所述的抗体，其特征在于，所述抗体选自：The antibody according to claim 1 or 2, wherein the antibody is selected from:
    (1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1、7、12、14或16所示的HCDR1，和/或包含SEQ ID NO:2、8、11、13、15或17所示的HCDR2，和/或包含SEQ ID NO:3或9任一所示的HCDR3；(1) an antibody comprising a heavy chain variable region, the heavy chain variable region comprising HCDR1 shown in SEQ ID NO: 1, 7, 12, 14 or 16, and/or comprising HCDR2 shown in SEQ ID NO: 2, 8, 11, 13, 15 or 17, and/or comprising HCDR3 shown in any of SEQ ID NO: 3 or 9;
    (2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:4所示的LCDR1，和/或包含SEQ ID NO:5所示的LCDR2，和/或包含SEQ ID NO:6或10任一所示的LCDR3；(2) an antibody comprising a light chain variable region comprising LCDR1 shown in SEQ ID NO:4, and/or comprising LCDR2 shown in SEQ ID NO:5, and/or comprising LCDR3 shown in any of SEQ ID NO:6 or 10;
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；(3) an antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody;
    (4)抗体，(1)～(3)中任一项所述抗体的变体，且具备与(1)～(3)中任一项所述抗体相同或相似的活性。(4) An antibody, a variant of the antibody described in any one of (1) to (3), having the same or similar activity as the antibody described in any one of (1) to (3).
  4. 如权利要求3所述的抗体，其特征在于，所述抗体选自：The antibody of claim 3, wherein the antibody is selected from:
    (1)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或 (1) an antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6; or
    (2)抗体，其包含SEQ ID NO:7所示的HCDR1，SEQ ID NO:8所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(2) an antibody comprising HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10;
    (3)抗体，其包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:11所示的HCDR2和SEQ ID NO:3所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3；或(3) an antibody comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:11 and HCDR3 shown in SEQ ID NO:3; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6;
    (4)抗体，其包含SEQ ID NO:12所示的HCDR1，SEQ ID NO:13所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(4) an antibody comprising HCDR1 shown in SEQ ID NO:12, HCDR2 shown in SEQ ID NO:13, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:10; or
    (5)抗体，其包含SEQ ID NO:14所示的HCDR1，SEQ ID NO:15所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；或(5) an antibody comprising HCDR1 shown in SEQ ID NO:14, HCDR2 shown in SEQ ID NO:15, and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:10; or
    (6)抗体，其包含SEQ ID NO:16所示的HCDR1，SEQ ID NO:17所示的HCDR2和SEQ ID NO:9所示的HCDR3；SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:10所示的LCDR3；(6) an antibody comprising HCDR1 shown in SEQ ID NO:16, HCDR2 shown in SEQ ID NO:17 and HCDR3 shown in SEQ ID NO:9; LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:10;
    (7)抗体，(1)～(6)中任一项所述抗体的变体，且具备与(1)～(6)中任一项所述抗体相同或相似的活性。(7) An antibody, a variant of the antibody described in any one of (1) to (6), having the same or similar activity as the antibody described in any one of (1) to (6).
  5. 如权利要求1-4中任一项所述的抗体，其特征在于，所述抗体选自：The antibody according to any one of claims 1-4, wherein the antibody is selected from:
    (1)抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:18、22、26、28、30或32所示的氨基酸序列、或上述序列的变体或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；(1) an antibody comprising a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence shown in SEQ ID NO: 18, 22, 26, 28, 30 or 32, or a variant of the above sequence or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence;
    (2)抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:20或24所示的氨基酸序列、或上述序列的变体或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；(2) an antibody comprising a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20 or 24, or a variant of the above sequence or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the above sequence;
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。(3) An antibody comprising (1) the heavy chain variable region of the antibody and (2) the light chain variable region of the antibody.
  6. 如权利要求5所述的抗体，其特征在于，所述抗体选自：The antibody of claim 5, wherein the antibody is selected from:
    (1)抗体，所述抗体的重链可变区具有SEQ ID NO:18所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列； (1) an antibody, the heavy chain variable region of the antibody has an amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence, and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 20 or has 90%, 91%, 92%, 93%, 94%, 95% identity with the above sequence, Amino acid sequences with 96%, 97%, 98%, 99% identity;
    (2)抗体，所述抗体的重链可变区具有SEQ ID NO:22所示的氨基酸序列或与上述序列具有95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；(2) an antibody, the heavy chain variable region of the antibody has an amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence with 95%, 96%, 97%, 98%, 99% identity with the above sequence, and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 24 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence amino acid sequence;
    (3)抗体，所述抗体的重链可变区具有SEQ ID NO:26所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:20所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；(3) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 26 or has an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20 or has 90%, 91%, 92%, 93%, 94%, 95% identity with the above sequence %, 96%, 97%, 98%, 99% identity amino acid sequence;
    (4)抗体，所述抗体的重链可变区具有SEQ ID NO:28所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；(4) an antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 28 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence with the above sequence, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or has 90%, 91%, 92%, 93%, 94%, 95% with the above sequence %, 96%, 97%, 98%, 99% identity amino acid sequence;
    (5)抗体，所述抗体的重链可变区具有SEQ ID NO:30所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；(5) Antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 30 or has the amino acid sequence with the above sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or has the amino acid sequence with the above sequence of 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99% identity amino acid sequence;
    (6)抗体，所述抗体的重链可变区具有SEQ ID NO:32所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列，所述轻链可变区具有SEQ ID NO:24所示的氨基酸序列或与上述序列具有90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列。(6) Antibody, the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO: 32 or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence with the above sequence, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or has 90%, 91%, 92%, 93%, 94%, 95% with the above sequence %, 96%, 97%, 98%, 99% identity amino acid sequence.
    (7)抗体，(1)～(6)中任一项所述抗体的变体，且具备与(1)～(6)中任一项所述抗体相同或相似的活性。(7) An antibody, a variant of the antibody described in any one of (1) to (6), having the same or similar activity as the antibody described in any one of (1) to (6).
  7. 如权利要求1-6任一项所述的抗体，其特征在于，所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体或IgG4抗体。The antibody according to any one of claims 1-6, wherein the antibody is a full antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment, multifunctional antibody or IgG4 antibody.
  8. 如权利要求1-7任一所述的抗体，其特征在于，所述抗体不显著结合NKG2C、NKG2E或其组合。The antibody of any one of claims 1-7, wherein the antibody does not significantly bind to NKG2C, NKG2E or a combination thereof.
  9. 如权利要求1-8任一所述的抗体，其特征在于，所述抗体结合NKG2A/CD94，不显著结合NKG2C/CD94、NKG2E/CD94或其组合；和/或，The antibody according to any one of claims 1-8, wherein the antibody binds to NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94 or a combination thereof; and/or,
    所述抗体结合表达NKG2A/CD94的细胞，不显著结合表达NKG2C/CD94、NKG2E/CD94或其组合的细胞。 The antibodies bind to cells expressing NKG2A/CD94 and do not significantly bind to cells expressing NKG2C/CD94, NKG2E/CD94, or combinations thereof.
  10. 如权利要求1-9任一所述的抗体，其特征在于，所述抗体在降低CD94/NKG2A介导的抑制表达CD94/NKG2A的细胞毒性淋巴细胞的细胞毒性中更有效。The antibody according to any one of claims 1-9, wherein the antibody is more effective in reducing CD94/NKG2A-mediated cytotoxicity of cytotoxic lymphocytes expressing CD94/NKG2A.
  11. 如权利要求10所述的抗体，其特征在于，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞、NKT细胞、α/βT细胞或γ/δT细胞。The antibody according to claim 10, wherein the cytotoxic lymphocytes expressing CD94/NKG2A are NK cells, NKT cells, α/βT cells or γ/δT cells.
  12. 如权利要求10所述的抗体，其特征在于，所述表达CD94/NKG2A的细胞毒性淋巴细胞是NK细胞。The antibody of claim 10, wherein the cytotoxic lymphocytes expressing CD94/NKG2A are NK cells.
  13. 一种免疫辍合物，其特征在于，所述免疫辍合物包括：权利要求1-12任一所述的抗体，以及与之连接的功能性分子。An immune conjugate, characterized in that the immune conjugate comprises: the antibody according to any one of claims 1-12, and a functional molecule linked thereto.
  14. 嵌合受体，其特征在于，所述嵌合受体的胞外域包含权利要求1-12任一所述的抗体，所述嵌合受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。A chimeric receptor, characterized in that the extracellular domain of the chimeric receptor comprises the antibody of any one of claims 1-12, and the chimeric receptor is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC) or a combination thereof.
  15. 如权利要求14所述嵌合受体，其特征在于，所述嵌合受体包含顺序连接的如下结构区域：权利要求1-12任一所述的抗体、跨膜区和胞内信号区。The chimeric receptor according to claim 14, characterized in that the chimeric receptor comprises the following structural regions connected in sequence: the antibody according to any one of claims 1-12, the transmembrane region and the intracellular signal region.
  16. 如权利要求15所述的嵌合受体，所述的胞内信号区选自：CD3δ、FcεRIγ、CD27、CD28、CD137、CD134、MyD88、CD40的胞内信号区序列或其组合；和/或The chimeric receptor according to claim 15, wherein the intracellular signal region is selected from the group consisting of the intracellular signal region sequences of CD3δ, FcεRIγ, CD27, CD28, CD137, CD134, MyD88, CD40 or combinations thereof; and/or
    所述的跨膜区包含CD8或CD28的跨膜区。The transmembrane region includes the transmembrane region of CD8 or CD28.
  17. 如权利要求16所述的嵌合受体，其特征在于，所述嵌合受体选自如下中的任意：The chimeric receptor of claim 16, wherein the chimeric receptor is selected from any of the following:
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区和CD3δ；或The antibody of any one of claims 1-12, the transmembrane region of CD8/CD28 and CD3δ; or
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区、CD137的胞内信号区和CD3δ；或The antibody of any one of claims 1-12, the transmembrane region of CD8/CD28, the intracellular signal region of CD137 and CD3δ; or
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区和CD3δ；或The antibody of any one of claims 1-12, the transmembrane region of CD8/CD28, the intracellular signal region of CD28 and CD3δ; or
    权利要求1-12任一所述的抗体、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3δ。The antibody of any one of claims 1-12, the transmembrane region of CD8/CD28, the intracellular signal region of CD28, CD137 and CD3δ.
  18. 如权利要求14所述的嵌合受体，其特征在于，所述嵌合受体的氨基酸序列如SEQ ID NO：115或116所示。The chimeric receptor according to claim 14, wherein the amino acid sequence of the chimeric receptor is as shown in SEQ ID NO: 115 or 116.
  19. 一种生物材料，其为如下中的任意一种：A biological material that is any of the following:
    1)编码权利要求1-12任一所述的抗体、权利要求13所述的免疫缀合物、权利要求14-18任一所述的嵌合受体的核酸；1) nucleic acid encoding the antibody according to any one of claims 1-12, the immunoconjugate according to claim 13, or the chimeric receptor according to any one of claims 14-18;
    2)包含1)所述的表达载体；或者2) comprising the expression vector described in 1); or
    3)包含1)或2)所述的病毒。3) comprising the virus described in 1) or 2).
  20. 一种宿主细胞，其包含权利要求14-18任一所述的嵌合受体。A host cell comprising the chimeric receptor of any one of claims 14-18.
  21. 如权利要求20所述的宿主细胞，其特征在于，所述宿主细胞结合表达NKG2A/CD94的细胞，不显著结合NKG2C/CD94、NKG2E/CD94或其组合。 The host cell of claim 20, wherein the host cell binds to cells expressing NKG2A/CD94 and does not significantly bind to NKG2C/CD94, NKG2E/CD94, or a combination thereof.
  22. 如权利要求20或21所述的宿主细胞，其特征在于，所述宿主细胞能抵抗NK细胞攻击或杀伤NK细胞。The host cell according to claim 20 or 21, wherein the host cell can resist NK cell attack or kill NK cell.
  23. 如权利要求20-22任一所述的宿主细胞，其特征在于，所述宿主细胞还表达识别肿瘤抗原和/或病原体抗原的嵌合受体。The host cell according to any one of claims 20-22, wherein the host cell also expresses a chimeric receptor that recognizes tumor antigens and/or pathogen antigens.
  24. 如权利要求20-22任一所述的宿主细胞，其特征在于，所述宿主细胞与靶向肿瘤和/或病原体的第二宿主细胞联合应用。The host cell according to any one of claims 20-22, wherein the host cell is used in combination with a second host cell targeting tumors and/or pathogens.
  25. 如权利要求20-24任一所述的宿主细胞，其特征在于，所述宿主细胞和/或第二宿主细胞不表达B2M、TCR/B2M、TCR/B2M/CIITA、TCR/B2M/NKG2A、和/或TCR/B2M/CIITA/NKG2A。The host cell according to any one of claims 20-24, wherein the host cell and/or the second host cell does not express B2M, TCR/B2M, TCR/B2M/CIITA, TCR/B2M/NKG2A, and/or TCR/B2M/CIITA/NKG2A.
  26. 如权利要求20-25任一所述的宿主细胞，其特征在于，所述宿主细胞和/或第二宿主细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、干细胞衍生的免疫效应细胞或其组合。The host cell according to any one of claims 20-25, wherein the host cell and/or the second host cell are T cells, natural killer cells, cytotoxic T lymphocytes, natural killer T cells, DNT cells, regulatory T cells, NK92 cells, stem cell-derived immune effector cells or a combination thereof.
  27. 如权利要求26所述的宿主细胞，其特征在于，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；The host cell according to claim 26, wherein the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells;
    优选地，所述T细胞为自体/同种异体T细胞；Preferably, the T cells are autologous/allogeneic T cells;
    优选地，所述T细胞为原代T细胞；Preferably, the T cells are primary T cells;
    优选地，所述T细胞来源于人的自体T细胞。Preferably, the T cells are derived from human autologous T cells.
  28. 联合用药，其特征在于，权利要求1-12任一所述的抗体、权利要求13所述的免疫缀合物、权利要求14-18任一所述的嵌合受体、权利要求20-27任一所述的宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联用；Combination medicine, characterized in that the antibody according to any one of claims 1-12, the immunoconjugate according to claim 13, the chimeric receptor according to any one of claims 14-18, the host cell according to any one of claims 20-27 are administered in combination with agents that enhance their functions, preferably in combination with chemotherapy drugs;
    和/或与改善其相关的一种或多种副作用的药剂联合施用；and/or in combination with agents that ameliorate one or more side effects associated therewith;
    和/或与表达靶向NKG2A之外的嵌合受体的宿主细胞联合施用。And/or in combination with host cells expressing chimeric receptors targeting other than NKG2A.
  29. 一种药物组合物，其特征在于，其包括：A pharmaceutical composition, characterized in that it comprises:
    权利要求1-12任一所述的抗体或编码该抗体的核酸；或The antibody of any one of claims 1-12 or a nucleic acid encoding the antibody; or
    权利要求13所述的免疫缀合物或编码该免疫缀合物的核酸；或The immunoconjugate of claim 13 or a nucleic acid encoding the immunoconjugate; or
    权利要求14-18任一所述的嵌合受体或编码该嵌合受体的核酸；或The chimeric receptor of any one of claims 14-18 or a nucleic acid encoding the chimeric receptor; or
    权利要求20-27任一所述的宿主细胞；The host cell according to any one of claims 20-27;
    以及药学上可接受的载体或赋形剂。and a pharmaceutically acceptable carrier or excipient.
  30. 一种试剂盒，其特征在于，其包括：A test kit, characterized in that it comprises:
    容器，以及位于容器中的权利要求30所述的药物组合物；或 a container, and the pharmaceutical composition of claim 30 in the container; or
    容器，以及位于容器中的权利要求1-12任一所述的抗体或编码该抗体的核酸；或权利要求13所述的免疫缀合物或编码该免疫缀合物的核酸；或权利要求14-18任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或权利要求20-27任一所述的宿主细胞。 A container, and the antibody of any one of claims 1-12 or the nucleic acid encoding the antibody in the container; or the immunoconjugate of claim 13 or the nucleic acid encoding the immunoconjugate; or the chimeric antigen receptor of any one of claims 14-18 or the nucleic acid encoding the chimeric antigen receptor; or the host cell of any one of claims 20-27.
PCT/CN2023/073176 2022-01-24 2023-01-19 Nkg2a antibody and application thereof WO2023138661A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153995A (en) * 2018-11-07 2020-05-15 上海怀越生物科技有限公司 NKG2A antibody, and preparation method and application thereof
CN113316590A (en) * 2018-11-16 2021-08-27 百时美施贵宝公司 anti-NKG 2A antibodies and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111153995A (en) * 2018-11-07 2020-05-15 上海怀越生物科技有限公司 NKG2A antibody, and preparation method and application thereof
CN113316590A (en) * 2018-11-16 2021-08-27 百时美施贵宝公司 anti-NKG 2A antibodies and uses thereof

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